Methods and materials relating to novel CD39-like polypeptides

ABSTRACT

The invention provides novel polynucleotides isolated from cDNA libraries of human fetal liver-spleen and macrophage as well as polypeptides encoded by these polynucleotides and mutants or variants thereof. The polypeptides correspond to a novel human CD39-like protein. Other aspects of the invention include vectors containing polynucleotides of the invention, and related host cells as well a processes for producing novel CD39-like polypeptides, and antibodies specific for such polypeptides.

1. RELATED APPLICATIONS

[0001] This patent application is a continuation-in-part of PCT patentapplication Serial No. PCT/US99/16180 [Attorney Docket No. 28110/35836]filed Jul. 16, 1999 which is a continuation-in-part of U.S. patentapplication Ser. No. 09/350836 [Attorney Docket No. 28110/35761] filedJul. 9, 1999 which is a continuation-in-part of U.S. patent applicationSer. No. 09/273,447 filed Mar. 19, 1999 which is a continuation-in-partof U.S. patent application Ser. No. 09/122,449 filed Jul. 24, 1998 andalso a continuation-in-part of U.S. patent application Ser. No.09/244,444 filed Feb. 4, 1999, which in turn is a continuation of U.S.patent application Ser. No. 09/118,205 filed Jul. 16, 1998, thedisclosures of all of which are incorporated by reference herein intheir entirety.

2. FIELD OF THE INVENTION

[0002] This invention relates in general to novel polynucleotidesisolated from cDNA libraries of human fetal liver-spleen and macrophagesand to polypeptides encoded by these polynucleotides. In particular, theinvention relates to a human CD39-like protein with homologies to ATPdiphosphohydrolases and variants thereof.

3. BACKGROUND

[0003] CD39 (cluster of differentiation 39) is a cell-surface moleculerecognized by a “cluster” of monoclonal antibodies that can be used toidentify the lineage or stage of differentiation of lymphocytes and thusto distinguish one class of lymphocytes from another. This CD39 moleculewas originally defined as a B lymphocyte marker (Rowe, M., et al. Int.J. Cancer 29:373 (1982)). Subsequent studies have shown CD39 to be amarker for a distinct subset of activated lymphocytes within theallosensitized CD8-positive cytotoxic cells (Gouttefangeas C., et al.,Eur. J.Immunol. 22:2681 (1992)). Outside of lymphoid tissue, CD39 can befound in quiescent vascular endothelial cells (Kansas, G. S., et al., J.Immunol. 146:2235 (1991)) and throughout rat brain in the neurons of thecerebral cortex, hippocampus, and cerebellum, as well as in glial cells(Wang, T-F. and Guidotti, G., Brain Res. 790:318 (1998)).

[0004] CD39 is a 510-amino acid protein with a predicted molecular massof 57 kDa. However, because of heavy glycosylation at asparagineresidues (six potential N-glycosylation sites) the molecule displays amobility closer to 100 kDa (Maliszewski, C. R., et al., J. Immunol.153:3574 (1994)). CD39 contains two hydrophobic regions, one near theamino terminus and the other near the carboxyl terminus which arebelieved to be transmembrane regions.

[0005] The role of CD39 in platelet aggregation and ATP/ADP hydrolysisis unclear. Although CD39 was originally reported to be an ectoADPasewith a preference for ATP over ADP as a substrate, Wang, et al., J.Biol. Chem. 271:9898-9901(1996), Marcus, et al., J. Clin. Invest.99:1351-1360 (1997) reported that CD39 was unique for its highpreference for ADP over ATP as a substrate and in 1998, Gayle, et al.,J. Clin Invest. 10:1851-1859 (1998), described CD39 as an ectoADPasewith no preference for one substrate over the other.

[0006] Reports that several ATP Diphosphohydrolases (ATPDases) shareamino acid sequence homology with CD39 have been substantiated by theshowing that CD39 is itself an ATPDase (Wang, T- F., et al., J. Biol.Chem. 271:9898 (1996); Kaczmarek, E., et al., J. Biol. Chem. 271:33116(1996)). Since CD39 is a plasma membrane-bound enzyme, CD39 has beentermed an “ecto-ATPase,” but CD39 is more often referred to as an“ecto-apyrase” because of the reduced rate of hydrolysis of ADP whencompared with ecto-ATPases.

[0007] This activity has shown to modulate platelet reactivity andaggregation in response to vascular injury. During vascular injury,activated platelets aggregate forming an occlusive thrombus. Excessiveplatelet accumulation at sites of vascular injury can contribute tovessel occlusion. Endothelial cells respond to the potentially occlusiveeffects of platelet aggregation by several mechanisms. One of thesemechanisms results ecto-apyrase-mediated removal of ADP, which in turneliminates platelet reactivity and recruitment. It is now known that theendothelial ecto-apyrase responsible for this ADP removal is CD39(Marcus, A. J., et al., J. Clin. Invest. 99:1351 (1997)).

[0008] Recently, CD39 was engineered to produce a soluble form of themolecule. This soluble CD39 was shown to display the same nucleotidaseactivity as the membrane-bound molecule (Gayle, R. B., et al., J. Clin.Invest. 101:1851 (1998)). Intravenously administered soluble CD39 alsoremained active in mice for an extensive period of time, indicating thatsoluble CD39 could be useful as a inhibitor of platelet aggregation inthe prophylaxis or treatment of platelet-mediated thrombotic conditions.

[0009] Platelet aggregation inhibitors (antithrombotic agents) decreasethe formation or the action of chemical signals that promote plateletaggregation. Currently available antithrombotic agents include aspirin,ticlopidine, and dipyridamole. These agents have proven beneficial inthe prevention and treatment of occlusive cardiovascular diseases,including myocardial infarction, cerebral ischemia, angina.Antithrombotic therapy has also been used in the maintenance of vasculargrafts.

[0010] Myocardial infarction is the development of necrosis of themyocardium (the middle muscular layer of the heart wall) due to acritical imbalance between oxygen and myocardial demand. The most commoncause of acute myocardium infarction is narrowing of the epicardialblood vessels due to atheromatous plaques. Plaque rupture withsubsequent exposure of basement membrane results in platelet aggregationand thrombus formation, which can result in partial or completeocclusion of the vessel and subsequent myocardial ischemia.

[0011] In cerebral ischemia, inadequate blood flow results from anocclusion in a blood vessel or hemorrhaging. In the latter case,excessive bleeding in one area of the brain deprives another area ofblood. If the damage occurs in a singular small area, “transient” or“focused” cerebral ischemia results. When a major artery is blocked(carotid artery) global or diffused ischemia results. The primarymedical strategy for secondary prevention of stroke is antiplatelettherapy. Aspirin is currently employed for reducing the risk ofrecurrent transient ischemic attacks or stroke in men who have transientischemia of the brain due to fibrin emboli.

[0012] Each year, thousands of patients suffer a decline in blood flowto one or more limbs. Without sufficient blood flow, and, unless bloodflow can be restored in time, the limb must be amputated. In some cases,grafts from the patient's veins can be used to form new arteries.However, in cases where the quality of the veins is poor, polymericvascular grafts are typically used. The polymeric grafts are inherentlythrombogenic as the blood constituents passing through the grafts becomeactivated and tend to form clots. Efforts to line the grafts withendothelial cells can reduce blood clotting, but better results areobtained when antithrombotic therapy is employed.

[0013] Angina pectoris is a characteristic chest pain caused byinadequate blood flow through the blood vessels of the myocardium. Theimbalance between oxygen delivery and utilization may result from aspasm of the vascular smooth muscle or from obstruction of blood vesselscaused by atherosclerotic lesions. Three classes of drugs have beenshown to be effective in treating angina: nitrates, beta-blockers andcalcium channel blockers. Currently, the antithrombotics dipyridamoleand aspirin are employed to prophylactically treat angina pectoris.

[0014] Ecto-apyrases, such as CD39, offer a number of advantages overseveral of the standard antithrombotics. For example, aspirin treatmentcontrols the prothrombotic action of thromboxane; however, aspirin alsoprevents formation of antithrombotic prostacyclin, which limitsaspirin's efficacy. Another antithrombotic, endothelium-derived relaxingfactor (nitric oxide; “EDRF/NO”), is inhibited in vitro and in vivo byhemoglobin after its rapid diffusion into erythrocytes. In contrast,CD39 is aspirin-insensitive and completely inhibits platelet reactivityeven when eicosanoid and EDRF/NO production are blocked.

[0015] CD39's ATPDase activity also implicates CD39 in the modulation ofneurotransmission. ATP is a major purinergic neurotransmitter that isoften co-released into the synaptic cleft with severalneurotransmitters. Responses to ATP are mediated by specific plasmamembrane receptors, called P2 purinergic receptors (Dubyak, G. R. andEl-Motassim,C. Am J. Physiol. 34:C577-C606 (1993)). The distribution ofCD39 in the rat brain indicates that CD39 plays a role in terminating P2purinergic neurotransmission (Wang, T. F. and Guidotti, G., Brain Res.790:318 (1998)). Furthermore, a decrease in ecto-apyrase activity isbelieved to lead to an accumulation of the excitatory neurotransmitter,extracellular ATP, as well as a deficiency of the endogenousanticonvulsant extracellular adenosine.

[0016] The chomosomal localization of CD39 provides additional supportfor a role in modulation of neurotransmission. More specifically, CD39has been mapped to chromosome 10q 23.1-24.1 (Maliszewski, C. R., et al.,J. Immunol. 153:3574 (1994)), and this site overlaps with thesusceptibility locus for human partial epilepsy with audiogenic symptoms(Ottman, R. et al., Nature Genet. 10:56 (1995)). This co-localization ofthe CD39 gene and the susceptibility locus has led to the hypothesisthat decrease in ecto-apyrase activity in the brain is the primary causeof partial epilepsy (Wang T-F., et al., Mol. Brain Res. 47:295 (1997)).

[0017] A screen for human cDNAs that hybridize to cosmids from the humanchromosome 9q34 region lead to the identification of a transcript withhigh homology to a chicken muscle ecto-ATPase (60% identity) and theecto-apyrase CD39 (41% amino acid identity) (Chadwick, B. P., Mamm.Genome 8.668 (1997)). This gene, designated “CD39-like-1 gene” (CD39L1),has a higher degree of homology to CD39 than does chicken muscleecto-ATPase. The biological activity of this protein has not been testedbut on the basis of the high amino acid homology, CD39L1 is believed tobe a new member of the ecto-ATPase family. Recently, a mouse gene withhomology to NTPases was cloned and sequenced (Acc. No. AF006482) byChadwick et al. (Mamm. Gen. 9:162-164 (1998).)

4. SUMMARY OF THE INVENTION

[0018] The invention is based on polynucleotides isolated from cDNAlibraries prepared from human fetal liver-spleen and macrophages. Thecompositions of the present invention include novel isolatedpolypeptides with apyrase and/or NDPase activity, in particular, novelhuman CD39-like polypeptides, and active variants thereof, isolatedpolynucleotides encoding such polypeptides, including recombinant DNAmolecules, cloned genes or degenerate variants thereof, especiallynaturally occurring variants such as allelic variants, antisensepolynucleotide molecules, and antibodies that specifically recognize oneor more epitopes present on such polypeptides, as well as hybridomasproducing such antibodies.

[0019] The compositions of the invention additionally include vectors,including expression vectors, containing the polynucleotides of theinvention, cells genetically engineered to contain such polynucleotidesand cells genetically engineered to express such polynucleotides.

[0020] The isolated polynucleotides of the invention include naturallyoccurring or wholly or partially synthetic DNA, e.g., cDNA and genomicDNA and RNA, e.g., mRNA. One polynucleotide according to the inventionencodes a novel CD39-like protein having the amino acid sequence shownin FIG. 2 (SEQ ID NO. 3), which has been designated CD39-L4. Anotherpolynucleotide according to the invention encodes a novel CD39-likeprotein having the amino acid sequence shown in SEQ ID NO: 27, which hasbeen designated CD39-L2. In another embodiment, a polynucleotideaccording to the invention encodes a novel CD39-like protein having thefull length or mature amino acid sequence set forth in SEQ ID NO. 25,which has been designated CD39-L66, and is an isoform of CD39-L4. Theisolated polynucleotides of the invention include a polynucleotidecomprising the nucleotide sequence of SEQ ID NO. 2, 24 or 26. Thepolynucleotides of the invention also include polynucleotides thatencode polypeptides with a biological activity of the polypeptide of SEQID NO. 3 or 27 (including apyrase or NDPase activity) such as (a) thenucleotide sequence of SEQ ID NO. 2, 24, 26 or (b) a nucleotide sequenceencoding the full length or mature amino acid sequence of SEQ ID NO. 3,25, or 27; (c) a polynucleotide which is an allelic variant of anypolynucleotide recited above; (d) a polynucleotide that hybridizes understringent conditions to (a) or (b); (e) or a polynucleotide that encodesa polypeptide comprising at least one CD39-like domain, e.g. catalyticdomain.

[0021] The polynucleotides of the invention additionally include thecomplement of any of the polynucleotides recited above.

[0022] The invention also provides a polynucleotide including anucleotide sequence that is substantially equivalent to thesepolynucleotides. Polynucleotides according to the invention can have atleast about 80%, more typically at least about 90%, and even moretypically at least about 95%, sequence identity to a polynucleotide ofSEQ ID NO. 2, 24 or 26 and specifically include a human polynucleotidewhich has at least 80% sequence identity to a polynucleotide of SEQ IDNO. 2, 24 or 26; or a polynucleotide which has at least 90% sequenceidentity to a polynucleotide of SEQ ID NO. 2, 24 or 26 . Similarly,polypeptides of the invention include polypeptides having apyrase orNDPase activity and at least about 80%, 90% or 95% sequence identity toSEQ ID NO. 3, 25 or 27.

[0023] A further aspect of the invention is the development of novelCD39-L4 variants which preferably have improved ADPase or NDPaseactivity compared to wild type CD39-L4 (SEQ ID NO: 5). This aspect ofthe invention includes polypeptides comprising at least one amino acidsubstitution selected from the group consisting of: D168-T, S170-Q andL175-F, wherein said substitution(s) result in increased ADPase activityof the polypeptide. One preferred embodiment is the polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 7 (encoded by thenucleotide sequence of SEQ ID NO. 6), which is a variant CD39-L4containing all three substitutions that has been designated ACRIII. Aplasmid containing this DNA was deposited with the American Type CultureCollection (ATCC), 10801 University Avenue, Manassas, Va., on Jul. 13,1999 under the terms of the Budapest Treaty (ATCC accession number______). Alternatively, instead of making the specific D168-T, S170-Qand/or L175-F substitution(s), substitution of amino acids with similarproperties is contemplated. Additional conservative substitutions atamino acid positions other than D168, S170 and/or L175 are furthercontemplated. For example, all of the corresponding amino acids fromCD39 could be substituted for amino acids 167-181 of CD39-L66 orCD39-L4.

[0024] In addition, development of novel CD39-L2 variants whichpreferably have improved ADPase or NDPase activity compared to wild typeCD39-L2 (SEQ ID NO: 27) is also contemplated. This aspect of theinvention includes polypeptides comprising at least one amino acidsubstitution wherein said substitution(s) result in increased ADPaseactivity of the polypeptide.

[0025] Polynucleotides encoding these polypeptides, vectors and hostcells comprising such polynucleotides, methods of using such host cellsto produce polypeptides, and other therapeutic products comprising thepolypeptides (including fusion proteins in which the CD39-likepolypeptide is fused to a heterologous peptide or polypeptide, such asan immunoglobulin constant region, or derivatives in which the CD39-likepolypeptide is modified by water soluble polymers to increase itshalf-life) are also comprehended by the invention, as are methods oftreating a subject suffering from a disorder relating to thrombosis,coagulation or platelet aggregation by administering such therapeuticproducts.

[0026] The invention further comprises methods of inhibiting plateletaggregation in a mammalian subject by reducing the ratio of ADP:ATP in amammalian subject to a less than normal ratio by administering thepolypeptides of the invention or by administering polypeptides withADPase activty and at least about 90% sequence identity to SEQ ID NO: 3,25 or 27. Preferably the ratio of ADP:ATP is reduced withoutsignificantly affecting ATP levels. In one embodiment, the ADP:ATP ratiois reduced systemically in circulation. In another embodiment, theADP:ATP ratio is reduced locally, for example, in heart, brain, kidney,lungs, limbs or other organs.

[0027] Methods of identifying compounds capable of reducing the ratio ofADP:ATP to a less than normal ratio are also contemplated. For example,compounds may be identified by steps including: determining apyraseactivity of said compound on ATP; determining apyrase activity of saidcompounds on ADP; and selecting a compound that has greater activitywith respect to ADP compared to ATP. Exemplary compounds to be screenedinclude, but are not limited to, CD39-L4 and CD39-L2 variants.

[0028] Gene therapy techniques are also provided to modulate diseasestates associated with CD39-L4 or CD39-L2 expression and/or biologicalactivity. Delivery of a functional CD39-L4 or CD39-L2 gene toappropriate cells is effected ex vivo, in situ, or in vivo by use ofvectors, and more particularly viral vectors (e.g., adenovirus,adeno-associated virus, or a retrovirus), or ex vivo by use of physicalDNA transfer methods (e.g., liposomes or chemical treatments).

[0029] The invention also relates to methods for producing polypeptidesof the invention comprising growing a culture of cells of the inventionin a suitable culture medium under conditions permitting expression ofthe desired polypeptide, and purifying the protein from the cells or theculture medium. Preferred embodiments include those in which the proteinproduced by such process is a mature form of the protein.

[0030] Protein compositions of the present invention, includingtherapeutic compositions, comprise polypeptides of the invention andoptionally an acceptable carrier, such as a hydrophilic (e.g.,pharmaceutically acceptable) carrier.

[0031] Polynucleotides according to the invention have numerousapplications in a variety of techniques known to those skilled in theart of molecular biology. These techniques include use as hybridizationprobes, use as oligomers for PCR, use for chromosome and gene mapping,use in the recombinant production of protein, and use in generation ofanti-sense DNA or RNA, their chemical analogs and the like. For example,because the expression of CD39-L4 and CD39-L2 mRNA is largely restrictedto specific tissues (CD39-L4 in macrophages and CD39-L2 in adult heartand fetal brain), polynucleotides of the invention can be used ashybridization probes to detect the presence of specific mRNA in a sampleusing, e.g., in situ hybridization.

[0032] In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath, et al.,Science 258:52-59 (1992), as expressed sequence tags for physicalmapping of the human genome.

[0033] A polynucleotide according to the invention can be joined to anyof a variety of other nucleotide sequences by well-establishedrecombinant DNA techniques (see Sambrook, J., et al. (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Usefulnucleotide sequences for joining to polypeptides include an assortmentof vectors, e.g., plasmids, cosmids, lambda phage derivatives,phagemids, and the like, that are well known in the art. Accordingly,the invention also provides a vector including a polynucleotide of theinvention and a host cell containing the polynucleotide. In general, thevector contains an origin of replication functional in at least oneorganism, convenient restriction endonuclease sites, and a selectablemarker for the host cell. Vectors according to the invention includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors. A host cell according to the invention can be aprokaryotic or eukaryotic cell and can be a unicellular organism or partof a multicellular organism.

[0034] The polypeptides according to the invention can be used in avariety of conventional procedures and methods that are currentlyapplied to other proteins. For example, a polypeptide of the inventioncan be used to generate an antibody which specifically binds thepolypeptide. The polypeptides of the invention having ATPDase activityare also useful for inhibiting platelet aggregation and can therefore beemployed in the prophylaxis or treatment of pathological conditionscaused by the inflammatory response. The polypeptides of the inventioncan also be used as molecular weight markers, and as a food supplement.

[0035] Another aspect of the invention is an antibody that specificallybinds the polypeptide of the invention. Such antibodies can be eithermonoclonal or polyclonal antibodies, as well fragments thereof andhumanized forms or fully human forms, such as those produced intransgenic animals. The invention further provides a hybridoma thatproduces an antibody according to the invention and anti-idiotypeantibodies.

[0036] Antibodies of the invention are useful for detection and/orpurification of the polypeptides of the invention.

[0037] Methods are also provided for preventing, treating orameliorating a medical condition, including thrombotic diseases, whichcomprises administering to a mammalian subject, including but notlimited to humans, a therapeutically effective amount of a compositioncomprising a polypeptide of the invention or a therapeutically effectiveamount of a composition comprising a binding partner of (e.g., antibodyspecifically reactive for) CD39-like polypeptides of the invention. Themechanics of the particular condition or pathology will dictate whetherthe polypeptides of the invention or binding partners (or inhibitors) ofthese would be beneficial to the individual in need of treatment.

[0038] The invention also provides a method of inhibiting plateletfunction comprising administering a CD39-L4 or CD39-L2 polypeptide ofthe invention to a medium comprising platelets. According to thismethod, polypeptides of the invention can be administered to produce anin vitro or in vivo inhibition of platelet function. A polypeptide ofthe invention can be administered in vivo as antithrombotic agent aloneor as an adjunct to other therapies.

[0039] Also provided are methods of hydrolyzing nucleotidediphosphatescomprising administering CD39-L4 or CD39-L2 polypeptides of theinvention to a medium comprising nucleotidediphosphates. According tothis method, polypeptides of the invention can be administered toproduce an in vitro or in vivo hydrolysis of nucleotidediphosphates. Apolypeptide of the invention can be administered in vivo alone or as anadjunct to other therapies.

[0040] The invention further provides methods for manufacturingmedicaments useful in the above described methods relating to plateletaggregation and thrombosis.

[0041] The invention also provides methods for detecting or quantitatingthe presence of the polynucleotides or polypeptides of the invention ina tissue or fluid sample, and corresponding kits that comprise suitablepolynucleotide probes or antibodies, together with an optionalquantitative standard. Such methods and kits can be utilized as part ofprognostic and diagnostic evaluation of patients and for theidentification of subjects exhibiting a predisposition to plateletmediated conditions.

[0042] The invention also provides methods for the identification ofcompounds that modulate (i.e. increase or decrease) the expression oractivity of the polynucleotides and/or polypeptides of the invention.Such methods can be utilized, for example, for the identification ofcompounds and other substances that interact with (e.g., bind to) thepolypeptides of the invention, and assays for identifying compounds andother substances that enhance or inhibit the activity of thepolypeptides of the invention, such assays comprising the step ofmeasuring activity of such polypeptides in the presence and absence ofthe test compound.

5. BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 shows polynucleotide sequences according to the invention.SEQ ID NO:1 was obtained from the b2HFLS20W cDNA library using standardPCR, sequencing by hybridization signature analysis, and single pass gelsequencing technology. A- adenosine; C-cytosine; G-guanosine; T-thymine.Ambiguous positions are designated as follows: R indicates A or G; Mindicates A or C; W indicates A or T; Y indicates C or T; S indicates Cor G; K indicates G or T; V indicates A or C or G; H indicates A or C orT; D indicates A or G or T; B indicates C or G or T; and N indicates anyof the four bases.

[0044] SEQ ID NO:2 is an extended version of SEQ ID NO:1 which wasobtained as described in Example 2.

[0045]FIG. 2 shows an amino acid sequence corresponding to thepolynucleotide sequence of SEQ ID NO:2. This sequence is designated asSEQ ID NO:3. The open reading frame encoding SEQ ID NO:3 begins atnucleotide 246 (numbered from the 5′ end) of SEQ ID NO:2. A- Alanine;R-Arginine; N- Asparagine; D- Aspartic Acid; C- Cysteine; E- GlutamicAcid; Q-Glutamine; G- Glycine; H- Histidine; I- Isoleucine; L- Leucine;K- Lysine; M-Methionine; F- Phenylalanine; P- Proline; S- Serine; T-Threonine; W-Tryptophan; Y- Tyrosine; V- Valine; X - any of the twentyamino acids.

[0046]FIG. 3 shows the amino acid sequence alignment of SEQ ID NO:3(identified as “246 prot”) and human CD39 (“CD39Human.seq”). The aminoacid residues are designated as for FIG. 2. The alignment was generatedusing the Jotun Hein method with the PAM250 residue weight table. Gapsare indicated by dashes; residues that are identical between the twosequences (within 1 distance unit) are boxed.

[0047]FIG. 4 shows the amino acid sequence alignment of SEQ ID NO:3(identified as “264 prot”) and murine NTPase (“mur ntpase”). The aminoacid residues are designated as for FIG. 2. The alignment was generatedas discussed for FIG. 3 Gaps are indicated by dashes; residues that areidentical between the two sequences (within 1 distance unit) are boxed.

[0048]FIG. 5 shows the apyrase conserved regions (ACR) in CD39-L4 inbold. ACR I starts at Phe 53, ACR II starts at Pro 124 and ACR IIIstarts at Met 167. The boxed sections highlight the amino acidsubstitutions that were made in the wild type CD39-L4 amino acidsequence to form mutants designated ACRI, ACRII and ACRIII.

[0049]FIG. 6 (SEQ ID NOS: 6 and 7) shows the nucleotide andcorresponding amino acid sequences of a preferred ACRIII mutantcontaining the following substitutions in the wild type CD39-L4 aminoacid sequence: D168-T, S170-Q and L175-F.

[0050]FIG. 7 shows the ADPase activity of CD39-L4 variants ACRI, ACRIIand ACRIII in comparison to wild type CD39-L4: (1) CD39-L4 ACR I mutant;(2) CD39-L4 ACR II mutant; (3) CD39-L4 ACR III mutant; (4) CD39-L4 wildtype; (5) sCD39; and (6) pSecTag2 vector (Invitrogen).

[0051]FIG. 8 shows the amino acid sequence alignment of SEQ ID NO. 3,SEQ ID NO. 25 (previously identified as SEQ ID NO. 5 in FIG. 5 of U.S.Ser. No. 09/122,449) and human CD39 (“CD39Human.seq”). The alignment wasgenerated using the Jotun Hein method with the PAM250 residue weighttable. Gaps are indicated by dashes; residues that are identical betweenthe two sequences (within 1 distance unit) are boxed.

[0052]FIG. 9 shows the amino acid sequence alignment of SEQ ID NO. 3,SEQ ID NO. 25 (previously identified as SEQ ID NO. 5 in FIG. 6 of U.S.Ser. No. 09/122,449) and the murine NTPase (“mur ntpase”). The alignmentwas generated as discussed for FIG. 8. Gaps are indicated by dashes;residues that are identical between the two sequences (within 1 distanceunit) are boxed.

6. DETAILED DESCRIPTION

[0053] 6.1 Definitions

[0054] The term “nucleotide sequence” refers to a heteropolymer ofnucleotides or the sequence of these nucleotides. The terms “nucleicacid” and “polynucleotide” are also used interchangeably herein to referto a heteropolymer of nucleotides. Generally, nucleic acid segmentsprovided by this invention may be assembled from fragments of the genomeand short oligonucleotide linkers, or from a series of oligonucleotides,to provide a synthetic nucleic acid which is capable of being expressedin a recombinant transcriptional unit comprising regulatory elementsderived from a microbial or viral operon.

[0055] An “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” is a stretch of polypeptide nucleotide residueswhich is long enough to use in polymerase chain reaction (PCR) orvarious hybridization procedures to identify or amplify identical orrelated parts of mRNA or DNA molecules.

[0056] “Oligonucleotides” or “nucleic acid probes” are prepared based onthe cDNA sequence provided in the present invention. Oligonucleotidescomprise portions of the DNA sequence having at least about 15nucleotides and usually at least about 20 nucleotides. Nucleic acidprobes comprise portions of the sequence having fewer nucleotides thanabout 6 kb, usually fewer than about 1 kb. After appropriate testing toeliminate false positives, these probes may be used to determine whethermRNAs are present in a cell or tissue or to isolate similar nucleic acidsequences from chromosomal DNA as described by Walsh, P. S., et al (1992PCR Methods Appl 1:241-250).

[0057] The term “probes” includes naturally occurring or recombinantsingle- or double-stranded nucleic acids or chemically synthesizednucleic acids. They may be labeled by nick translation, Klenow fill-inreaction, PCR or other methods well known in the art. Probes of thepresent invention, their preparation and/or labeling are elaborated inSambrook, J., et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, NY; or Ausubel, F. M., et al (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York N.Y., bothincorporated herein by reference.

[0058] The term “stringent” is used to refer to conditions that arecommonly understood in the art as stringent. An exemplary set ofconditions include a temperature of 60-70° C., (preferably about 65° C.)and a salt concentration of 0.70 M to 0.80 M (preferably about 0.75M).Further exemplary conditions include, hybridizing conditions that (1)employ low ionic strength and high temperature for washing, for example,0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employduring hybridization a denaturing agent such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

[0059] The term “recombinant,” as used herein, means that a polypeptideor protein is derived from recombinant (e.g., microbial or mammalian)expression systems. “Microbial” refers to recombinant polypeptides orproteins made in bacterial or fungal (e.g., yeast) expression systems.As a product, “recombinant microbial” defines a polypeptide or proteinessentially free of native endogenous substances and unaccompanied byassociated native glycosylation. Polypeptides or proteins expressed inmost bacterial cultures, e.g., E. coli, will be free of glycosylationmodifications; polypeptides or proteins expressed in yeast will have aglycosylation pattern different from that expressed in mammalian cells.

[0060] The term “recombinant expression vehicle or vector” refers to aplasmid or phage or virus or vector, for expressing a polypeptide from aDNA (RNA) sequence. The expression vehicle can comprise atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structural or coding sequence which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription initiation and termination sequences. Structural unitsintended for use in yeast or eukaryotic expression systems preferablyinclude a leader sequence enabling extracellular secretion of translatedprotein by a host cell. Alternatively, where recombinant protein isexpressed without a leader or transport sequence, it may include anN-terminal methionine residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal product.

[0061] “Recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.The cells can be prokaryotic or eukaryotic. Recombinant expressionsystems as defined herein will express heterologous polypeptides orproteins upon induction of the regulatory elements linked to the DNAsegment or synthetic gene to be expressed.

[0062] The term “open reading frame,” ORF, means a series of tripletscoding for amino acids without any termination codons and is a sequencetranslatable into protein.

[0063] The term “expression modulating fragment,” EMF, means a series ofnucleotide molecules which modulates the expression of an operablylinked ORF or EMF. As used herein, a sequence is said to “modulate theexpression of an operably linked sequence” when the expression of thesequence is altered by the presence of the EMF. EMFs include, but arenot limited to, promoters, and promoter modulating sequences (inducibleelements). One class of EMFs are fragments which induce the expressionof an operably linked ORF in response to a specific regulatory factor orphysiological event.

[0064] As used herein, an “uptake modulating fragment,” UMF, means aseries of nucleotide molecules which mediate the uptake of a linked DNAfragment into a cell. UMFs can be readily identified using known UMFs asa target sequence or target motif with the computer-based systems knownin the art.

[0065] The presence and activity of a UMF can be confirmed by attachingthe suspected UMF to a marker sequence. The resulting nucleic acidmolecule is then incubated with an appropriate host under appropriateconditions and the uptake of the marker sequence is determined. Asdescribed above, a UMF will increase the frequency of uptake of a linkedmarker sequence.

[0066] “Active” refers to those forms of the polypeptide which retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide.

[0067] “Naturally occurring polypeptide” refers to polypeptides producedby cells that have not been genetically engineered and specificallycontemplates various polypeptides arising from post-translationalmodifications of the polypeptide including, but not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidationand acylation.

[0068] “Derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

[0069] “Recombinant variant” refers to any polypeptide differing fromnaturally occurring polypeptides by amino acid insertions, deletions,and substitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as cellular trafficking,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology.

[0070] Preferably, amino acid “substitutions” are the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, ora threonine with a serine, i.e., conservative amino acid replacements.“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a polypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity.

[0071] As used herein, “substantially equivalent” can refer both tonucleotide and amino acid sequences, for example a mutant sequence, thatvaries from a reference sequence by one or more substitutions,deletions, or additions, the net effect of which does not result in anadverse functional dissimilarity between the reference and subjectsequences. Typically, such a mutant sequence varies from one of thoselisted herein by no more than about 20% (i.e., the number ofsubstitutions, additions, and/or deletions in a mutant sequence, ascompared to the corresponding listed sequence, divided by the totalnumber of residues in the mutant sequence is about 0.2 or less). Such amutant sequence is said to have 80% sequence identity to the listedsequence. In one embodiment, a mutant sequence of the invention variesfrom a listed sequence by no more than 10% (90% sequence identity), in avariation of this embodiment, by no more than 5% (95% sequenceidentity), and in a further variation of this embodiment, by no morethan 2% (98% sequence identity). Mutant amino acid sequences accordingto the invention generally have at least 95% sequence identity with alisted amino acid sequence, whereas mutant nucleotide sequence of theinvention can have lower percent sequence identities. For the purposesof the present invention, sequences having substantially equivalentbiological activity and substantially equivalent expressioncharacteristics are considered substantially equivalent. For thepurposes of determining equivalence, truncation of the mature sequenceshould be disregarded.

[0072] Where desired, an expression vector may be designed to contain a“signal or leader sequence” which will direct the polypeptide throughthe membrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

[0073] A polypeptide “fragment,” “portion,” or “segment” is a stretch ofamino acid residues of at least about 5 amino acids, often at leastabout 7 amino acids, typically at least about 9 to 13 amino acids, and,in various embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to display biologicand/or immunologic activity.

[0074] Alternatively, recombinant variants encoding these same orsimilar polypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polypeptide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

[0075] “Activated” cells as used in this application are those which areengaged in extracellular or intracellular membrane trafficking,including the export of neurosecretory or enzymatic molecules as part ofa normal or disease process.

[0076] The term “purified” as used herein denotes that the indicatednucleic acid or polypeptide is present in the substantial absence ofother biological macromolecules, e.g., polynucleotides, proteins, andthe like. In one embodiment, the polynucleotide or polypeptide ispurified such that it constitutes at least 95% by weight, morepreferably at least 99.8% by weight, of the indicated biologicalmacromolecules present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 1000daltons, can be present).

[0077] The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

[0078] The term “infection” refers to the introduction of nucleic acidsinto a suitable host cell by use of a virus or viral vector.

[0079] The term “transformation” means introducing DNA into a suitablehost cell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

[0080] The term “transfection” refers to the taking up of an expressionvector by a suitable host cell, whether or not any coding sequences arein fact expressed.

[0081] The term “intermediate fragment” means a nucleic acid between 5and 1000 bases in length, and preferably between 10 and 40 bp in length.

[0082] Each of the above terms is meant to encompasses all that isdescribed for each, unless the context dictates otherwise.

[0083] 6.2 Hybridization Conditions

[0084] Suitable hybridization conditions may be routinely determined byoptimization procedures or pilot studies. Such procedures and studiesare routinely conducted by those skilled in the art to establishprotocols for use in a laboratory. See e.g., Ausubel, et al., CurrentProtocols in Molecular Biology, Vol. 1-2, John Wiley & Sons (1989);Sambrook, et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Vols.1-3, Cold Springs Harbor Press (1989); and Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold SpringHarbor, N.Y. (1982), all of which are incorporated by reference herein.For example, conditions such as temperature, concentration ofcomponents, hybridization and washing times, buffer components, andtheir pH and ionic strength may be varied.

[0085] 6.3 Nucleic Acids of the Invention

[0086] The sequences falling within the scope of the present inventionare not limited to the specific sequences herein described, but alsoinclude allelic variations thereof. Allelic variations can be routinelydetermined by comparing the sequence provided in SEQ ID NOs: 1, 2, 24 or26, a representative fragment thereof, or a nucleotide sequence at least99.9% identical to SEQ ID NO: 1, 2, 24 or 26 with a sequence fromanother isolate of the same species. Furthermore, to accommodate codonvariability, the invention includes nucleic acid molecules coding forthe same amino acid sequences as do the specific ORFs disclosed herein.In other words, in the coding region of an ORF, substitution of onecodon for another which encodes the same amino acid is expresslycontemplated.

[0087] Any specific sequence disclosed herein can be readily screenedfor errors by resequencing a particular fragment, such as an ORF, inboth directions (i.e., sequence both strands).

[0088] The present invention further provides recombinant constructscomprising a nucleic acid having the sequence of any one of SEQ ID NO:1, 2, 24 or 26, the mature protein coding sequence or a fragmentthereof. The recombinant constructs of the present invention comprise avector, such as a plasmid or viral vector, into which a nucleic acidhaving the sequence of any one of SEQ ID NO: 1, 2, 24 or 26 or afragment thereof is inserted, in a forward or reverse orientation. Inthe case of a vector comprising one of the ORFs of the presentinvention, the vector may further comprise regulatory sequences,including for example, a promoter, operably linked to the ORF. Forvectors comprising the EMFs and UMFs of the present invention, thevector may further comprise a marker sequence or heterologous ORFoperably linked to the EMF or UMF. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTl, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

[0089] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

[0090] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0091] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0092] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0093] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced or derepressed by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

[0094] Included within the scope of the nucleic acid sequences of theinvention are nucleic acid sequences that hybridize under stringentconditions to a fragment of the DNA sequences in FIG. 1, which fragmentis greater than about 10 bp, preferably 20-50 bp, and even greater than100 bp, including 200 bp or greater, 300 bp or greater, 400 bp orgreater, and 500 bp or greater.

[0095] In accordance with the invention, polynucleotide sequences whichencode the novel nucleic acids, or functional equivalents thereof, maybe used to generate recombinant DNA molecules that direct the expressionof that nucleic acid, or a functional equivalent thereof, in appropriatehost cells.

[0096] The nucleic acid sequences of the invention are further directedto sequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. The amino acid sequence variants of the nucleic acids arepreferably constructed by mutating the polynucleotide to give an aminoacid sequence that does not occur in nature. These amino acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite.

[0097] Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

[0098] In a preferred method, polynucleotides encoding the novel nucleicacids are changed via site-directed mutagenesis. This method usesoligonucleotide sequences that encode the polynucleotide sequence of thedesired amino acid variant, as well as a sufficient adjacent nucleotideon both sides of the changed amino acid to form a stable duplex oneither side of the site being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982).

[0099] PCR may also be used to create amino acid sequence variants ofthe novel nucleic acids. When small amounts of template DNA are used asstarting material, primer(s) that differs slightly in sequence from thecorresponding region in the template DNA can generate the desired aminoacid variant. PCR amplification results in a population of product DNAfragments that differ from the polynucleotide template encoding thepolypeptide at the position specified by the primer. The product DNAfragments replace the corresponding region in the plasmid and this givesthe desired amino acid variant.

[0100] A further technique for generating amino acid variants is thecassette mutagenesis technique described in Wells et al., Gene 34:315(1985); and other mutagenesis techniques well known in the art, such as,for example, the techniques in Sambrook, et al., supra, and CurrentProtocols in Molecular Biology, Ausubel, et al.

[0101] Due to the inherent degeneracy of the genetic code, other DNAsequences which encode substantially the same or a functionallyequivalent amino acid sequence may be used in the practice of theinvention for the cloning and expression of these novel nucleic acids.Such DNA sequences include those which are capable of hybridizing to theappropriate novel nucleic acid sequence under stringent conditions.

[0102] Furthermore, knowledge of the DNA sequence provided by thepresent invention allows for the modification of cells to permit, orincrease, expression of endogenous CD39-like polypeptides. Cells can bemodified (e.g., by homologous recombination) to provide increasedCD39-like expression by replacing, in whole or in part, the naturallyoccurring CD39-like promoter with all or part of a heterologous promoterso that the cells express CD39-like polypeptides at a higher level. Theheterologous promoter is inserted in such a manner that it isoperatively linked to CD39-like encoding sequences. See, for example,PCT International Publication No. WO94/12650, PCT InternationalPublication No. WO92/20808, and PCT International Publication No.WO91/09955. It is also contemplated that, in addition to heterologouspromoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and themultifunctional CAD gene which encodes carbamyl phosphate synthase,aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may beinserted along with the heterologous promoter DNA. If linked to theCD39-like coding sequence, amplification of the marker DNA by standardselection methods results in co-amplification of the CD39-like codingsequences in the cells.

[0103] The polynucleotides of the present invention also make possiblethe development, through, e.g., homologous recombination or knock outstrategies, of animals that fail to express functional CD39-likepolypeptides or that express a variant of a CD39-like polypeptide. Suchanimals are useful as models for studying the in vivo activities ofCD39-like polypeptides as well as for studying modulators of CD39-likepolypeptides.

[0104] 6.4 Identification of Polymorphisms

[0105] Polymorphisms can be identified in a variety of ways known in theart which all generally involve obtaining a sample from a patient,analyzing DNA from the sample, optionally involving isolation oramplification of the DNA, and identifying the presence of thepolymorphism in the DNA. For example, PCR may be used to amplify anappropriate fragment of genomic DNA which may then be sequenced.Alternatively, the DNA may be subjected to allele-specificoligonucleotide hybridization (in which appropriate oligonucleotides arehybridized to the DNA under conditions permitting detection of a singlebase mismatch) or to a single nucleotide extension assay (in which anoligonucleotide that hybridizes immediately adjacent to the position ofthe polymorphism is extended with one or more labelled nucleotides). Inaddition, traditional restriction fragment length polymorphism analysis(using restriction enzymes that provide differential digestion of thegenomic DNA depending on the presence or absence of the polymorphism)may be performed.

[0106] Alternatively, a polymorphism resulting in a change in the aminoacid sequence could also be detected by detecting a corresponding changein amino acid sequence of the protein, e.g., by an antibody specific tothe variant sequence.

[0107] 6.5 Hosts

[0108] The present invention further provides host cells containing SEQID NO: 1, 2, 24 or 26 of the present invention, wherein the nucleic acidhas been introduced into the host cell using known transformation,transfection or infection methods. The host cell can be a highereukaryotic host cell, such as a mammalian cell, a lower eukaryotic hostcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the recombinant construct intothe host cell can be effected by calcium phosphate transfection, DEAE,dextran mediated transfection, or electroporation (Davis, L., et al.,Basic Methods in Molecular Biology (1986)).

[0109] The host cells containing one of SEQ ID NO: 1, 2, 24 or 26 of thepresent invention, can be used in conventional manners to produce thegene product encoded by the isolated fragment (in the case of an ORF) orcan be used to produce a heterologous protein under the control of theEMF.

[0110] Any host/vector system can be used to express one or more of theORFs of the present invention. These include, but are not limited to,eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9cells, as well as prokaryotic host such as E. coli and B. subtilis. Themost preferred cells are those which do not normally express theparticular polypeptide or protein or which expresses the polypeptide orprotein at low natural level.

[0111] Mature proteins can be expressed in mammalian cells, yeast,bacteria, insect cells or other cells under the control of appropriatepromoters. Cell-free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

[0112] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

[0113] Recombinant polypeptides and proteins produced in bacterialculture are usually isolated by initial extraction from cell pellets,followed by one or more salting-out, aqueous ion exchange or sizeexclusion chromatography steps. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

[0114] 6.6 Peptides

[0115] The present invention further provides isolated polypeptidesencoded by the nucleic acid fragments of the present invention or bydegenerate variants of the nucleic acid fragments of the presentinvention. Fragments may be fused to carrier molecules such asimmunoglobulins for many purposes, including increasing the valency ofprotein binding sites. For example, fragments of the protein may befused through “linker” sequences to the Fc portion of an immunoglobulin.For a bivalent form of the protein, such a fusion could be to the Fcportion of an IgG molecule. Other immunoglobulin isotypes may also beused to generate such fusions. For example, a protein-IgM fusion wouldgenerate a decavalent form of the protein of the invention. Analogs ofthe polypeptides of the invention can be fused to another moiety ormoieties, e,g., targeting moiety or another therapeutic agent. Suchanalogs may exhibit improved properties such as activity and/orstability. By “degenerate variant” is intended nucleotide fragmentswhich differ from a nucleic acid fragment of the present invention(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of thegenetic code, encode an identical polypeptide sequence. Preferrednucleic acid fragments of the present invention are the ORFs whichencode proteins.

[0116] The invention also provides both full length and mature forms(for example, without a signal sequence or precursor sequence) ofCD39-like polypeptides. The full length form of such proteins isidentified in the sequence listing by translation of the nucleotidesequence of each disclosed clone. The mature form of such protein may beobtained by expression of the full-length polynucleotide in a suitablemammalian cell or other host cell. The sequence of the mature form ofthe protein is also determinable from the amino acid sequence of thefull length form.

[0117] A variety of methodologies known in the art can be utilized toobtain any one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. This isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. In an alternative method, thepolypeptide or protein is purified from bacterial cells which naturallyproduce the polypeptide or protein. One skilled in the art can readilyfollow known methods for isolating polypeptides and proteins in order toobtain one of the isolated polypeptides or proteins of the presentinvention. These include, but are not limited to, immunochromatography,HPLC, size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel, et al., CurrentProtocols in Molecular Biology.

[0118] The polypeptides and proteins of the present invention canalternatively be purified from cells which have been altered to expressthe desired polypeptide or protein. As used herein, a cell is said to bealtered to express a desired polypeptide or protein when the cell,through genetic manipulation, is made to produce a polypeptide orprotein which it normally does not produce or which the cell normallyproduces at a lower level. One skilled in the art can readily adaptprocedures for introducing and expressing either recombinant orsynthetic sequences into eukaryotic or prokaryotic cells in order togenerate a cell which produces one of the polypeptides or proteins ofthe present invention.

[0119] The purified polypeptides are used in in vitro binding assayswhich are well known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, forexample, small molecules, molecules from combinatorial libraries,antibodies or other proteins. The molecules identified in the bindingassay are then tested for antagonist or agonist activity in in vivotissue culture or animal models that are well known in the art. Inbrief, the molecules are titrated into a plurality of cell cultures oranimals and then tested for either cell/animal death or prolongedsurvival of the animal/cells.

[0120] In addition, the binding molecules may be complexed with toxins,e.g., ricin or cholera, or with other compounds that are toxic to cells.The toxin-binding molecule complex is then targeted to the tumor orother cell by the specificity of the binding molecule for SEQ IDNOs:3-4.

[0121] 6.7 Gene Therapy

[0122] Mutations in the CD39-like gene that result in loss of normalfunction of the CD39-like gene product underlie CD39-related humandisease states. The invention comprehends gene therapy to restoreCD39-like activity that would thus be indicated in treating thosedisease states. Delivery of a functional CD39-like gene to appropriatecells is effected ex vivo, in situ, or in vivo by use of vectors, andmore particuarly viral vectors (e.g., adenovirus, adeno-associatedvirus, or a retrovirus), or ex vivo by use of physical DNA transfermethods (e.g., liposomes or chemical treatments). See, for example,Anderson, Nature, supplement to vol. 392, no 6679, pp. 25-30 (1998). Foradditional reviews of gene therapy technology, see Friedmann, Science,244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); andMiller, Nature, 357: 455-460 (1992). Alternatively, it is contemplatedthat in other human disease states, preventing the expression of orinhibiting the activity of CD39-like polypeptides will be useful intreating the disease states. It is contemplated that antisense therapyor gene therapy could be applied to negatively regulate the expressionof CD39-like polypeptides.

[0123] 6.8 Deposit of clone

[0124] A plasmid containing DNA encoding the ACR III mutant wasdeposited with the American Type Culture Collection (ATCC), 10801University Avenue, Manassas, Va., on Jul. 13, 1999 under the terms ofthe Budapest Treaty (ATCC accession no. ______).

[0125] 6.9 Antibodies

[0126] In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing the desiredantibody are well known in the art (Campbell, A. M., MonoclonalAntibodies Technology: Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands (1984); St. Groth, et al., J. Immunol. 35:1-21 (1990);Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique,the human B-cell hybridoma technique (Kozbor, et al., Immunology Today4:72 (1983); Cole, et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc. (1985), pp. 77-96). In addition, techniques forpreparing chimeric,and humanized antibodies (including polypeptidescontaining CDR and/or antigen-binding sequences of antibodies) are wellknown in the art.

[0127] Any animal (mouse, rabbit, etc.) which is known to produceantibodies can be immunized with a peptide or polypeptide of theinvention. Methods for immunization are well known in the art. Suchmethods include subcutaneous or intraperitoneal injection of thepolypeptide. One skilled in the art will recognize that the amount ofthe protein encoded by the ORF of the present invention used forimmunization will vary based on the animal which is immunized, theantigenicity of the peptide and the site of injection.

[0128] The protein which is used as an immunogen may be modified oradministered in an adjuvant in order to increase the protein'santigenicity. Methods of increasing the antigenicity of a protein arewell known in the art and include, but are not limited to, coupling theantigen with a heterologous protein (such as globulin or -galactosidase)or through the inclusion of an adjuvant during immunization.

[0129] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, such as SP2/0-Ag14myeloma cells, and allowed to become monoclonal antibody producinghybridoma cells.

[0130] Any one of a number of methods well known in the art can be usedto identify the hybridoma cell which produces an antibody with thedesired characteristics. These include screening the hybridomas with anELISA assay, western blot analysis, or radioimmunoassay (Lutz, et al.,Exp. Cell Research. 175:109-124 (1988)).

[0131] Hybridomas secreting the desired antibodies are cloned and theclass and subclass is determined using procedures known in the art(Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniquesin Biochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)).

[0132] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to proteins of the present invention.

[0133] For polyclonal antibodies, antibody containing antiserum isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures.

[0134] The present invention further provides the above-describedantibodies in detectably labeled form. Antibodies can be detectablylabeled through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase,alkaline phosphatase, etc.) fluorescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishingsuch labeling are well-known in the art, for example, see (Sternberger,L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. etal., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129(1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

[0135] The labeled antibodies of the present invention can be used forin vitro, in vivo, and in situ assays to identify cells or tissues inwhich a fragment of the polypeptide of interest is expressed. Theantibodies may also be used directly in therapies or other diagnostics.

[0136] The present invention further provides the above-describedantibodies immobilized on a solid support. Examples of such solidsupports include plastics such as polycarbonate, complex carbohydratessuch as agarose and sepharose, acrylic resins and polyacrylamide andlatex beads. Techniques for coupling antibodies to such solid supportsare well known in the art (Weir, D. M. et al., “Handbook of ExperimentalImmunology” 4th Ed., Blackwell Scientific Publications, Oxford, England,Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press,N.Y. (1974)). The immobilized antibodies of the present invention can beused for in vitro, in vivo, and in situ assays as well as forimmuno-affinity purification of the proteins of the present invention.

[0137] 6.10 Computer Readable Sequences

[0138] In one application of this embodiment, a nucleotide sequence ofthe present invention can be recorded on computer readable media. Asused herein, “computer readable media” refers to any medium which can beread and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention.

[0139] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention. A variety ofdata storage structures are available to a skilled artisan for creatinga computer readable medium having recorded thereon a nucleotide sequenceof the present invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. A skilled artisancan readily adapt any number of dataprocessor structuring formats (e.g.text file or database) in order to obtain computer readable mediumhaving recorded thereon the nucleotide sequence information of thepresent invention.

[0140] By providing the nucleotide sequence of SEQ ID NO: 1, 2, 24 or26, a representative fragment thereof, or a nucleotide sequence at least99.9% identical to SEQ ID NO: 1, 2, 24 or 26 in computer readable form,a skilled artisan can routinely access the sequence information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. Software which implements the BLAST (Altschul,et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag, et al.,Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system maybe used to identify open reading frames (ORFs) within a nucleic acidsequence. Such ORFs may be protein encoding fragments and may be usefulin producing commercially important proteins such as enzymes used infermentation reactions and in the production of commercially usefulmetabolites.

[0141] As used herein, “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate thatany one of the currently available computer-based systems is suitablefor use in the present invention.

[0142] As stated above, the computer-based systems of the presentinvention comprise a data storage means having stored therein anucleotide sequence of the present invention and the necessary hardwaremeans and software means for supporting and implementing a search means.As used herein, “data storage means” refers to memory which can storenucleotide sequence information of the present invention, or a memoryaccess means which can access manufactures having recorded thereon thenucleotide sequence information of the present invention.

[0143] As used herein, “search means” refers to one or more programswhich are implemented on the computer-based system to compare a targetsequence or target structural motif with the sequence information storedwithin the data storage means. Search means are used to identifyfragments or regions of a known sequence which match a particular targetsequence or target motif. A variety of known algorithms are disclosedpublicly and a variety of commercially available software for conductingsearch means are and can be used in the computer-based systems of thepresent invention. Examples of such software includes, but is notlimited to, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). Askilled artisan can readily recognize that any one of the availablealgorithms or implementing software packages for conducting homologysearches can be adapted for use in the present computer-based systems.

[0144] As used herein, a “target sequence” can be any nucleic acid oramino acid sequence of six or more nucleotides or two or more aminoacids. A skilled artisan can readily recognize that the longer a targetsequence is, the less likely a target sequence will be present as arandom occurrence in the database. The most preferred sequence length ofa target sequence is from about 10 to 100 amino acids or from about 30to 300 nucleotide residues. However, it is well recognized that searchesfor commercially important fragments, such as sequence fragmentsinvolved in gene expression and protein processing, may be of shorterlength.

[0145] As used herein, “a target structural motif,” or “target motif,”refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

[0146] 6.11 Expression Modulating Sequences

[0147] EMF sequences can be identified within a genome by theirproximity to the ORFs. An intergenic segment, or a fragment of theintergenic segment, from about 10 to 200 nucleotides in length, taken 5′from any ORF will modulate the expression of an operably linked 3′ ORFin a fashion similar to that found with the naturally linked ORFsequence. As used herein, an “intergenic segment” refers to thefragments of a genome which are between two ORF(S) herein described.Alternatively, EMFs can be identified using known EMFs as a targetsequence or target motif in the computer-based systems of the presentinvention.

[0148] The presence and activity of an EMF can be confirmed using an EMFtrap vector. An EMF trap vector contains a cloning site 5′ to a markersequence. A marker sequence encodes an identifiable phenotype, such asantibiotic resistance or a complementing nutrition auxotrophic factor,which can be identified or assayed when the EMF trap vector is placedwithin an appropriate host under appropriate conditions, As describedabove, an EMF will modulate the expression of an operably linked markersequence. A more detailed discussion of various marker sequences isprovided below. A sequence which is suspected of being an EMF is clonedin all three reading frames in one or more restriction sites upstreamfrom the marker sequence in the EMF trap vector. The vector is thentransformed into an appropriate host using known procedures and thephenotype of the transformed host is examined under appropriateconditions. As described above, an EMF will modulate the expression ofan operably linked marker sequence.

[0149] 6.12 Triplex Helix Formation

[0150] In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee, et al., Nucl. Acids Res. 6:3073(1979); Cooney, et al., Science 15241:456 (1988); and Dervan, et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)).

[0151] Triple helix- formation optimally results in a shut-off of RNAtranscription from DNA, while antisense RNA hybridization blockstranslation of an mRNA molecule into polypeptide. Both techniques havebeen demonstrated to be effective in model systems. Informationcontained in the sequences of the present invention is necessary for thedesign of an antisense or triple helix oligonucleotide.

[0152] 6.13 Diagnostic Assays and Kits

[0153] The present invention further provides methods to identify theexpression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention.

[0154] In detail, such methods comprise incubating a test sample withone or more of the antibodies or one or more of nucleic acid probes ofthe present invention and assaying for binding of the nucleic acidprobes or antibodies to components within the test sample.

[0155] Conditions for incubating a nucleic acid probe or antibody with atest sample vary. Incubation conditions depend on the format employed inthe assay, the detection methods employed, and the type and nature ofthe nucleic acid probe or antibody used in the assay. One skilled in theart will recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

[0156] The test samples of the present invention include cells, proteinor membrane extracts of cells, or biological fluids such as sputum,blood, serum, plasma, or urine. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is compatible with the systemutilized.

[0157] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0158] Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the probes or antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound probe or antibody.

[0159] In detail, a compartment kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe.

[0160] Types of detection reagents include labeled nucleic acid probes,labeled secondary antibodies, or in the alternative, if the primaryantibody is labeled, the enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody. One skilled in theart will readily recognize that the disclosed probes and antibodies ofthe present invention can be readily incorporated into one of theestablished kit formats which are well known in the art.

[0161] 6.14 Screening Assays

[0162] Using the isolated proteins of the present invention, the presentinvention further provides methods of obtaining and identifying agentswhich bind to a protein encoded by one of the ORFs from a nucleic acidwith a sequence of one of SEQ ID NO: 1, 2, 24 or 26, or to a nucleicacid with a sequence of one of SEQ ID NO: 1, 2, 24 or 26.

[0163] In detail, said method comprises the steps of: (a) contacting anagent with an isolated protein encoded by one of the ORFs of the presentinvention, or nucleic acid of the invention; and (b) determining whetherthe agent binds to said protein or said nucleic acid.

[0164] The agents screened in the above assay can be, but are notlimited to, peptides, carbohydrates, vitamin derivatives, or otherpharmaceutical agents. The agents can be selected and screened at randomor rationally selected or designed using protein modeling techniques.

[0165] For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention.

[0166] Alternatively, agents may be rationally selected or designed. Asused herein, an agent is said to be “rationally selected or designed”when the agent is chosen based on the configuration of the particularprotein. For example, one skilled in the art can readily adapt currentlyavailable procedures to generate peptides, pharmaceutical agents and thelike capable of binding to a specific peptide sequence in order togenerate rationally designed antipeptide peptides, for example seeHurby, et al., Application of Synthetic Peptides: Antisense Peptides,”In Synthetic Peptides, A User's Guide, W. H. Freeman, N.Y. (1992), pp.289-307, and Kaspczak, et al., Biochemistry 28:9230-8 (1989), orpharmaceutical agents, or the like.

[0167] In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF fore expression control.

[0168] One class of DNA binding agents are agents which contain baseresidues which hybridize or form a triple helix formation by binding toDNA or RNA. Such agents can be based on the classic phosphodiester,ribonucleic acid backbone, or can be a variety of sulfhydryl orpolymeric derivatives which have base attachment capacity.

[0169] Agents suitable for use in these methods usually contain 20 to 40bases and are designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee, et al., Nucl. AcidsRes. 6:3073 (1979); Cooney, et al., Science 241:456 (1988); and Dervan,et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano,J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).Triple helix- formation optimally results in a shut-off of RNAtranscription from DNA, while antisense RNA hybridization blockstranslation of an mRNA molecule into polypeptide. Both techniques havebeen demonstrated to be effective in model systems. Informationcontained in the sequences of the present invention is necessary for thedesign of an antisense or triple helix oligonucleotide and other DNAbinding agents.

[0170] Agents which bind to a protein encoded by one of the ORFs of thepresent invention can be used as a diagnostic agent, in the control ofbacterial infection by modulating the activity of the protein encoded bythe ORF. Agents which bind to a protein encoded by one of the ORFs ofthe present invention can be formulated using known techniques togenerate a pharmaceutical composition.

[0171] 6.15 Use of Nucleic Acids as Probes

[0172] Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from thenucleotide sequence of the SEQ ID NO: 1, 2, 24 or 26. Because thecorresponding gene is expressed in only one out of 18 tissues tested,namely macrophages, a hybridization probe derived from SEQ ID NO: 1, 2,24 or 26 can be used as an indicator of the presence of macrophage RNAin a sample. Any suitable hybridization technique can be employed, suchas, for example, in situ hybridization.

[0173] PCR as described U.S. Pat. Nos. 4,683,195 and 4,965,188 providesadditional uses for oligonucleotides based upon the nucleotidesequences. Such probes used in PCR may be of recombinant origin, may bechemically synthesized, or a mixture of both. The probe will comprise adiscrete nucleotide sequence for the detection of identical sequences ora degenerate pool of possible sequences for identification of closelyrelated genomic sequences.

[0174] Other means for producing specific hybridization probes fornucleic acids include the cloning of nucleic acid sequences into vectorsfor the production of mRNA probes. Such vectors are known in the art andare commercially available and may be used to synthesize RNA probes invitro by means of the addition of the appropriate RNA polymerase as T7or SP6 RNA polymerase and the appropriate radioactively labelednucleotides.

[0175] The nucleotide sequences may be used to construct hybridizationprobes for mapping their respective genomic sequences. The nucleotidesequence provided herein may be mapped to a chromosome or specificregions of a chromosome using well known genetic and/or chromosomalmapping techniques. These techniques include in situ hybridization,linkage analysis against known chromosomal markers, hybridizationscreening with libraries or flow-sorted chromosomal preparationsspecific to known chromosomes, and the like. The technique offluorescent in situ hybridization of chromosome spreads has beendescribed, among other places, in Verma, et al (1988) Human Chromosomes:A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescentin situ hybridization of chromosomal preparations and other physicalchromosome mapping techniques may be correlated with additional geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of anucleic acid on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals.

[0176] The nucleotide sequence may be used to produce purifiedpolypeptides using well known methods of recombinant DNA technology.Among the many publications that teach methods for the expression ofgenes after they have been isolated is Goeddel, (1990) Gene ExpressionTechnology, Methods and Enzymology, Vol 185, Academic Press, San Diego.Polypeptides may be expressed in a variety of host cells, eitherprokaryotic or eukaryotic. Host cells may be from the same species fromwhich a particular polypeptide nucleotide sequence was isolated or froma different species. Advantages of producing polypeptides by recombinantDNA technology include obtaining adequate amounts of the protein forpurification and the availability of simplified purification procedures.

[0177] Each sequence so obtained was compared to sequences in GenBankusing a search algorithm developed by Applied Biosystems andincorporated into the INHERIT™ 670 Sequence Analysis System. In thisalgorithm, Pattern Specification Language (developed by TRW Inc., LosAngeles, Calif.) was used to determine regions of homology. The threeparameters that determine how the sequence comparisons run were windowsize, window offset, and error tolerance. Using a combination of thesethree parameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions of homology from chance matches. Smith-Waterman alignments wereused to display the results of the homology search.

[0178] Peptide and protein sequence homologies were ascertained usingthe INHERIT™ 670 Sequence Analysis System in a way similar to that usedin DNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

[0179] Alternatively, BLAST, which stands for Basic Local AlignmentSearch Tool, is used to search for local sequence alignments (Altschul,S. F., (1993) J Mol Evol 36:290-300; Altschul, S. F., et al (1990) J MolBiol 215:403-10). BLAST produces alignments of both nucleotide and aminoacid sequences to determine sequence similarity. Because of the localnature of the alignments, BLAST is especially useful in determiningexact matches or in identifying homologs. Whereas it is ideal formatches which do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP).

[0180] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0181] In addition, BLAST analysis was used to search for relatedmolecules within the libraries of the LIFESEQ™ database. This process,an “electronic northern” analysis is analogous to northern blot analysisin that it uses one cellubrevin sequence at a time to search foridentical or homologous molecules at a set stringency. The stringency ofthe electronic northern is based on “product score”. The product scoreis defined as (% nucleotide or amino acid [between the query andreference sequences] in Blast multiplied by the % maximum possible BLASTscore [based on the lengths of query and reference sequences]) dividedby 100. At a product score of 40, the match will be exact within a 1-2%error; and at 70, the match will be exact. Homologous or relatedmolecules can be identified by selecting those which show product scoresbetween approximately 15 and 30.

[0182] 6.16 SEQ ID NOs:1-8, 23 - 24 and 26 - 27

[0183] Referring to FIG. 1, SEQ ID NO:1 is the nucleotide sequence of anexpressed sequence tag corresponding to a polynucleotide isolated from acDNA library of human fetal liver-spleen. SEQ ID NO:2 is an extendedversion of SEQ ID NO:1 obtained as described in Example 2, and theencoded polypeptide in SEQ ID NO: 3 is referred to herein as CD39-L4.SEQ ID NO:2 encodes a polypeptide having the amino acid sequence of SEQID NO:3 (shown in FIG. 2). The open reading frame corresponding to SEQID NO:3 starts at nucleotide 246, as numbered from the 5′ end of SEQ IDNO:2. This open reading frame encodes a polypeptide 428 amino acids inlength. The estimated molecular weight of the unglycosylated polypeptideis approximately 47.52 kDa.

[0184] Protein database searches with the BLAST algorithm indicate thatSEQ ID NO:3 is homologous to the CD39 family. FIG. 3 shows the aminoacid sequence alignment between SEQ ID NO:3 (identified as “246 prot”)and human CD39 (“CD39Human.seq”), indicating that the two sequencesshare 30% amino acid sequence identity. Moreover, a higher degree ofhomology between the apyrase conserved regions (Kaczmarek et al., J.Biol. Chem. 271:33116-33122 (1996) is observed. In particular, an almostperfect match to a putative ATP-binding region was found from aminoacids 54-58, DAGST (DAGSS in CD39). In addition, the DLGGASTQ motif(DLGGASTQ in CD39), which is very well conserved among ATPDases, isfound from amino acids 199-206 in SEQ ID NO:3. Other regions conservedin apyrases were found from amino acids 129-134, ATAGLR (ATAGMR in CD39)and from amino acids 169-173, GSDEG (GQEEG in CD39).

[0185] SEQ ID NO:3 differs from CD39 in that SEQ ID NO:3 contains ahydrophobic stretch of 22 amino acids at its amino terminus, which isindicative of a leader peptide. SEQ ID NO:3 also lacks the transmembranedomain found at the carboxyl terminus of CD39. These features indicatethat SEQ ID NO:3 is a soluble ATPDase.

[0186] SEQ ID NO:3 shares an even higher degree of homology (86%identity) with a murine NTPase, as shown in the amino acid sequencealignment presented in FIG. 4 (SEQ ID NO:3 is identified as “246 prot,”and mouse CD39 as “mur ntpase”).

[0187] The message encoding SEQ ID NO:3 is tightly regulated in atissue-specific manner. An expression study using a semiquantitativePCR/Southern blot approach revealed a significant level of expression inmacrophage. In contrast, human CD39 is expressed in tissues such asplacenta, lung, skeletal muscle, kidney, and heart.

[0188] SEQ ID NO: 4 is the polynucleotide sequence for CD39-L4. SEQ IDNO: 5 is the corresponding amino acid sequence.

[0189] SEQ ID NO: 6 is the polynucleotide sequence for a CD39-L4 variantdesignated ACRIII, wherein the following amino acid substitutions havebeen made: D168-T, S170-Q and L175-F; SEQ ID NO: 7 is the correspondingamino acid sequence.

[0190] SEQ ID NO: 8 is the genomic sequence for the human CD39-L4 gene;exons appear at nucleotides 1-288 (exon 1), 1281-1580 (exon 2),1820-1855 (exon 3) 2467-2555 (exon 4), 2863-2942 (exon 5), 3889-3950(exon 6), 4894-4995 (exon 7), 5847-5987 (exon 8), 6966-7138 (exon 9) and8556-9365 (exon 10).

[0191] SEQ ID NO: 24 is the polynucleotide sequence for a CD39-L4 splicevariant that creates an isoform designated CD39-L66. SEQ ID NO: 25 isthe corresponding amino acid sequence.

[0192] SEQ ID NO: 26 is the polynucleotide sequence for CD39-L2. SEQ IDNO: 27 is the corresponding amino acid sequence.

[0193] 6.17 Uses of Novel CD39-Like Polypeptides and Antibodies

[0194] Polypeptides of the invention having ATPDase, including NDPase,activity are useful for inhibiting platelet function and can thereforebe employed in the prophylaxis or treatment of pathological conditionscaused by or involving thrombosis or excessive coagulation or excessiveplatelet aggregation, such as myocardial infarction, cerebral ischemia,angina, and the like. Polypeptides of the invention can also be used inthe maintenance of vascular grafts. Platelet function can be measured byany of a number of standard assays, such as, for example, the plateletaggregation assay described in Example 5.

[0195] Such pathological conditions include conditions caused by orinvolving arterial thrombosis, such as coronary artery thrombosis andresulting myocardial infarction, cerebral artery thrombosis orintracardiac thrombosis (due to, e.g., atrial fibrillation) andresulting stroke, and other peripheral arterial thrombosis andocclusion; conditions associated with venous thrombosis, such as deepvenous thrombosis and pulmonary embolism; conditions associated withexposure of the patient's blood to a foreign or injured tissue surface,including diseased heart valves, mechanical heart valves, vasculargrafts, and other extracorporeal devices such as intravascular cannulas,vascular access shunts in hemodialysis patients, hemodialysis machinesand cardiopulmonary bypass machines; and conditions associated withcoagulapathies, such as hypercoagulability and disseminatedintravascular coagulopathy. Co-administration of other agents suitablefor treating the pathological condition, e.g., other anti-coagulationagents, is also contemplated.

[0196] In particular, variants like the ACRIII mutant described hereinare expected to be superior therapeutics for treating such pathologicalconditions because (1) ACRIII exhibits six-fold greater activitycompared to wild type CD39-L4, and (2) ACRIII, like CD39-L4, is uniquelyspecific for ADP and does not hydrolyze ATP. Thus, adverse side effectsfrom hydrolysis of circulating ATP are avoided.

[0197] For instance, ATP is known to act as an extracellular signal inmany tissues. In the heart, extracellular ATP modulates ionic processesand contractile function (for review see Burnstock, G.,Neuropharmacology 36:1127). Recently, it has been shown thatextracellular ATP markedly inhibits glucose transport in ratcardiomyocytes (Fisher, Y. et al., J. Biol. Chem. 274:755-761. Anothersource of extracellular ATP is that released from parenchymal cellsunder hypoxic or ischemic conditions (Skobel, E., and Kammermeier, H.Biochim. Biophys. Acta 1362:128-134). ATP is also involved in themodulation of anti-IgE-induced release of histamine from human lung mastcells (Schulman, E. S., et al., Am. J. Respir. Cell Mol. Biol.20:520-537).

[0198] Furthermore, the ability of CD39-L4 to hydrolyze NDPs other thanADP has implications outside the circulatory system. For instance, ithas been reported that UDP is the most potent agonist for the human P2Y₆receptor. Communi, et al., Bioch Bioph Res Com 222:303-308 (1996). Thisreceptor is expressed in several tissues including infiltrating T cellspresent in inflammatory bowel disease. Somers, et al., Lab Invest78:1375-1383 (1998). In this microenvironment, a molecule with theenzymatic properties of CD39-L4 could influence T cell responses bymodifying the extracellular half-life of UDP. Another role for CD39-L4has been suggested by the report that mouse CD39-L4 maps closely to alocus associated with audigenic brain seizures in mice. See Chadwick, etal., Genomics 50:357-367 (1998); Seyfried, et al., Genetics 99:117-126(1981). This locus, known as Asp-1, is thought to be linked or tocorrespond to a factor that influences Ca²⁺-ATPase activity. Neumann, etal., Behav. Genetics 20:307-323 (1990).

[0199] Additionally, the polypeptides of the invention can be used asmolecular weight markers, and as a food supplement. A polypeptideconsisting of SEQ ID NO:3, for example, has a molecular mass ofapproximately 47.52 kD in its unglycosylated form. Protein foodsupplements are well known and the formulation of suitable foodsupplements including polypeptides of the invention is within the levelof skill in the food preparation art.

[0200] The polypeptides of the invention are also useful for makingantibody substances that are specifically immunoreactive with CD39-likeproteins. Antibodies and portions thereof (e.g., Fab fragments) whichbind to the polypeptides of the invention can be used to identify thepresence of such polypeptides in a sample. For example, the level of thenative protein corresponding to SEQ ID NO:3 in a blood sample can bedetermined as an indication of vascular condition. Such determinationsare carried out using any suitable immunoassay format, and anypolypeptide of the invention that is specifically bound by the antibodycan be employed as a positive control.

[0201] Additionally, the polypeptides of the invention are useful formodulating the ratios of levels of adenosine molecules in vivo toregulate homeostasis. Adenosine diphosphate (ADP) is an agonist ofplatelet activation and aggregation. It has been demonstrated that theP2Y receptor (and others including P2T and P2Y1 and potentially others)transduces this signal. Adenosine triphosphate (ATP) also binds to thisreceptor, but acts as an antagonist. Therefore, the ratios of levels ofATP/ADP can significantly influence in vivo platelet activation andaggregation. Agents that specifically decrease levels of ADP not onlydecrease the amount of agonist available to signal, but also increasethe relative antagonistic effects of ATP, because of less competitionfor the common receptor.

[0202] The polypeptides of the invention are administered by any routethat delivers an effective dosage to the desired site of action. Thedetermination of a suitable route of administration and an effectivedosage for a particular indication is within the level of skill in theart. For treatment of vascular disease, polypeptides according to theinvention are generally administered intravenously. In vivo murinestudies with soluble human CD39 have shown that mice injectedintravenously with 50 mg recombinant soluble human CD39 in 100 mlsterile saline had biologically active CD39 in their sera for anextended period of time, with an elimination half-life of almost 2 days(Gayle, R. B., et al., J. Clinical Invest. 101:1851-1859(1998)).Suitable dosage ranges for the polypeptides of the invention can beextrapolated from these dosages or from similar studies in appropriateanimal models. Dosages can then be adjusted as necessary by theclinician to provide maximal therapeutic benefit.

[0203] 6.18 PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

[0204] A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be administered to a patient in need, by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s) at doses to treat or ameliorate a variety of disorders.Such a composition may also contain (in addition to protein and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF,Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.

[0205] The pharmaceutical composition may further contain other agentswhich either enhance the activity of the protein or compliment itsactivity or use in treatment. For example, CD39-L2 or CD39-L4 may beco-administered with platelet ADP receptor antagonists, e.g. ATPderivatives, ADP derivatives. Such additional factors and/or agents maybe included in the pharmaceutical composition to produce a synergisticeffect with protein of the invention, or to minimize side effects.Conversely, protein of the present invention may be included informulations of the particular cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent to minimize side effects of the cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent. A protein of the present invention may beactive in multimers (e.g., heterodimers or homodimers) or complexes withitself or other proteins. As a result, pharmaceutical compositions ofthe invention may comprise a protein of the invention in such multimericor complexed form.

[0206] Techniques for formulation and administration of the compounds ofthe instant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

[0207] In practicing the method of treatment or use of the presentinvention, a therapeutically effective amount of protein of the presentinvention is administered to a mammal having a condition to be treated.Protein of the present invention may be administered in accordance withthe method of the invention either alone or in combination with othertherapies such as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors.

[0208] 6.18.1. ROUTES OF ADMINISTRATION

[0209] Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.Administration ofprotein of the present invention used in the pharmaceutical compositionor to practice the method of the present invention can be carried out ina variety of conventional ways, such as oral ingestion, inhalation,topical application or cutaneous, subcutaneous, intraperitoneal,parenteral or intravenous injection. Intravenous administration to thepatient is preferred.

[0210] Alternately, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a arthritic joints or in fibrotic tissue, often in a depotor sustained release formulation. In order to prevent the scarringprocess frequently occurring as complication of glaucoma surgery, thecompounds may be administered topically, for example, as eyedrops.Furthermore, one may administer the drug in a targeted drugdelivery system, for example, in a liposome coated with a specificantibody, targeting, for example, arthritic or fibrotic tissue. Theliposomes will be targeted to and taken up selectively by the afflictedtissue.

[0211] 6.18.2. COMPOSITIONS/FORMULATIONS

[0212] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in a conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein of the present invention is administered orally, protein of thepresent invention will be in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carriersuch as a gelatin or an adjuvant. The tablet, capsule, and powdercontain from about 5 to 95% protein of the present invention, andpreferably from about 25 to 90% protein of the present invention. Whenadministered in liquid form, a liquid carrier such as water, petroleum,oils of animal or plant origin such as peanut oil, mineral oil, soybeanoil, or sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of protein of the present invention, andpreferably from about 1 to 50% protein of the present invention.

[0213] When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0214] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.Dragee cores are providedwith suitable coatings. For this purpose, concentrated sugar solutionsmay be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or dragee coatings foridentification or to characterize different combinations of activecompound doses.

[0215] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

[0216] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0217] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentratedsolutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0218] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

[0219] A pharmaceutical carrier for the hydrophobic compounds of theinvention is a cosolvent system comprising benzyl alcohol, a nonpolarsurfactant, a water-miscible organic polymer, and an aqueous phase. Thecosolvent system may be the VPD co-solvent system. VPD is a solution of3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPDD:5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides may substitute for dextrose.Alternatively,other delivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

[0220] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols. Many of the proteinase inhibitingcompounds of the invention may be provided as salts withpharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts are those salts which retain thebiological effectiveness and properties of the free acids and which areobtained by reaction with inorganic or organic bases such as sodiumhydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,triethanol amine and the like.

[0221] The pharmaceutical composition of the invention may be in theform of a complex of the protein(s) of present invention along withprotein or peptide antigens. The protein and/or peptide antigen willdeliver a stimulatory signal to both B and T lymphocytes. B lymphocyteswill respond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunoglobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

[0222] The amount of protein of the present invention in thepharmaceutical composition of the present invention will depend upon thenature and severity of the condition being treated, and on the nature ofprior treatments which the patient has undergone. Ultimately, theattending physician will decide the amount of protein of the presentinvention with which to treat each individual patient. Initially, theattending physician will administer low doses of protein of the presentinvention and observe the patient's response. Larger doses of protein ofthe present invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight. For compositions of the presentinvention which are useful for bone, cartilage, tendon or ligamentregeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein of theinvention which may also optionally be included in the composition asdescribed above, may alternatively or additionally, be administeredsimultaneously or sequentially with the composition in the methods ofthe invention. Preferably for bone and/or cartilage formation, thecomposition would include a matrix capable of delivering theprotein-containing composition to the site of bone and/or cartilagedamage, providing a structure for the developing bone and cartilage andoptimally capable of being resorbed into the body. Such matrices may beformed of materials presently in use for other implanted medicalapplications.

[0223] The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability. Presently preferredis a 50:50 (mole weight) copolymer of lactic acid and glycolic acid inthe form of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

[0224] A preferred family of sequestering agents is cellulosic materialssuch as alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins ofthe invention may be combined with other agents beneficial to thetreatment of the bone and/or cartilage defect, wound, or tissue inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), transforminggrowth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growthfactor (IGF).

[0225] The therapeutic compositions are also presently valuable forveterinary applications. Particularly domestic animals and thoroughbredhorses, in addition to humans, are desired patients for such treatmentwith proteins of the present invention. The dosage regimen of aprotein-containing pharmaceutical composition to be used in tissueregeneration will be determined by the attending physician consideringvarious factors which modify the action of the proteins, e.g., amount oftissue weight desired to be formed, the site of damage, the condition ofthe damaged tissue, the size of a wound, type of damaged tissue (e.g.,bone), the patient's age, sex, and diet, the severity of any infection,time of administration and other clinical factors. The dosage may varywith the type of matrix used in the reconstitution and with inclusion ofother proteins in the pharmaceutical composition. For example, theaddition of other known growth factors, such as IGF I (insulin likegrowth factor I), to the final composition, may also effect the dosage.Progress can be monitored by periodic assessment of tissue/bone growthand/or repair, for example, X-rays, histomorphometric determinations andtetracycline labeling.

[0226] Polynucleotides of the present invention can also be used forgene therapy. Such polynucleotides can be introduced either in vivo orex vivo into cells for expression in a mammalian subject.Polynucleotides of the invention may also be administered by other knownmethods for introduction of nucleic acid into a cell or organism(including, without limitation, in the form of viral vectors or nakedDNA). Cells may also be cultured ex vivo in the presence of proteins ofthe present invention in order to proliferate or to produce a desiredeffect on or activity in such cells. Treated cells can then beintroduced in vivo for therapeutic purposes.

[0227] 6.18.3. EFFECTIVE DOSAGE

[0228] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve its intended purpose. Morespecifically, a therapeutically effective amount means an amounteffective to prevent development of or to alleviate the existingsymptoms of the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Forexample, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of the C-proteinase activity). Suchinformation can be used to more accurately determine useful doses inhumans.

[0229] A therapeutically effective dose refers to that amount of thecompound that results in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1.Dosage amount and interval may be adjustedindividually to provide plasma levels of the active moiety which aresufficient to maintain the C-proteinase inhibiting effects, or minimaleffective concentration (MEC). The MEC will vary for each compound butcan be estimated from in vitro data; for example, the concentrationnecessary to achieve 50-90% inhibition of the C-proteinase using theassays described herein. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.However, HPLC assays or bioassays can be used to determine plasmaconcentrations.

[0230] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen which maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%.In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

[0231] The amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

[0232] 6.18.4. PACKAGING

[0233] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labelled for treatment of an indicatedcondition.

[0234] The present invention is illustrated in the following examples.Upon consideration of the present disclosure, one of skill in the artwill appreciate that many other embodiments and variations may be madein the scope of the present invention. Accordingly, it is intended thatthe broader aspects of the present invention not be limited to thedisclosure of the following examples.

EXAMPLE 1 Isolation of SEQ ID NO:1 From a cDNA Library of Human FetalLiver-Spleen

[0235] A plurality of novel nucleic acids were obtained from a b2HFLS20WcDNA library prepared from human fetal liver-spleen, as described inBonaldo et al., Genome Res. 6:791-806 (1996), using standard PCR,Sequencing by hybridization sequence signature analysis, and Sangersequencing techniques. The inserts of the library were amplified withPCR using primers specific for vector sequences flanking the inserts.These samples were spotted onto nylon membranes and interrogated witholigonucleotide probes to give sequence signatures. The clones wereclustered into groups of similar or identical sequences, and singlerepresentative clones were selected from each group for gel sequencing.The 5′ sequence of the amplified inserts was then deduced using thereverse M13 sequencing primer in a typical Sanger sequencing protocol.PCR products were purified and subjected to fluorescent dye terminatorcycle sequencing. Single-pass gel sequencing was done using a 377Applied Biosystems (ABI) sequencer. One of these inserts was identifiedas a novel sequence not previously obtained from this library and notpreviously reported in public databases. This sequence is shown in FIG.1 as SEQ ID NO:1.

EXAMPLE 2 Isolation of SEQ ID NO:2 and Determination of a NucleotideSequence Encoding a 428-Amino Acid Protein with Sequence Homology toCD39

[0236] The nucleotide sequence shown in FIG. 1, and labeled SEQ ID NO:2,encodes the translated amino acid sequence SEQ ID NO:3, which is shownin FIG. 2. The extended nucleotide sequence was obtained by isolatingcolonies generated from pools of clones from a human macrophage cDNAlibrary (Invitrogen, Cat. # A550-25). Briefly, the macrophage cDNAlibrary was plated on LB/Amp plates (containing 100 mg/ml ampicillin) ata density of about 40,000 colonies/plate. The colonies were lifted ontonitrocellulose filters and hybridized with a radiolabeled probegenerated from the original clone (i.e., SEQ ID NO:1).

[0237] That the identified clones corresponded to SEQ ID NOs:1 and 2 wasconfirmed by using gene-specific primers (5′-GCTACCTCACTTCCTTTGAG-3′[SEQ ID NO: 9] and 5′-CTGGCTGGTGAAGTTTTCCTC-3′ [SEQ ID NO: 10]) in aPCR-based assay. Then PCR using vector- and gene-specific primers wasemployed to amplify the 5′ portion of the cDNA. Nested primers were usedto generate sequence from the amplified product(s). Laser gene™ softwarewas used to edit and “contig” the partial sequences into a full-lengthsequence. As discussed above, the amino acid sequence has strikinghomology to CD39, which is involved in modulating platelet reactivityduring vascular inflammation. Based in part on the observed sequencesimilarity to CD39, the polypeptide encoded by SEQ ID NO: 2 wasdesignated CD39-L4.

EXAMPLE 3 A. Expression of SEQ ID NOS. 3 and 5 in COS-7 Cells

[0238] COS-7 cells were grown in DMEM (ATCC) and 10% fetal bovine serum(FBS) (Gibco) to 70% confluence. Prior to transfection the media waschanged to DMEM and 0.5% FCS. Cells were transfected with cDNAs for SEQID NOs. 3 and 5 or with pBGaI vector by the FuGENE-6 transfectionreagent (Boehringer). In summary, 4 μl of FuGENE6 was diluted in 100 μlof DMEM and incubated for 5 minutes. Then, this was added to 1 μg of DNAand incubated for 15 minutes before adding it to a 35 mm dish of COS-7cells. The COS-7 cells were incubated at 37° C. with 5% CO₂. After 24hours, media and cell lysates were collected, centrifuged and dialyzedagainst assay buffer (15 mM Tris pH 7.6, 134 mM NaCl, 5 mM glucose, 3 mMCaCl₂ and MgCl₂. More robust expression can be achieved using theprotocol described in Example 6 below.

B. Expression Study Using SEQ ID NO:2

[0239] The expression of SEQ.ID NO. 2 in various tissues was analyzedusing a semi-quantitative polymerase chain reaction-based technique.Human cDNA libraries were used as sources of expressed genes fromtissues of interest (adult brain, adult heart, adult kidney, adult lymphnode, adult liver, adult lung, adult ovary, adult placenta, adultspleen, adult testis, bone marrow, fetal kidney, fetal liver, fetalliver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage).Gene-specific primers (5′-GCTACCTCACTTCCTTTGAG-3′ [SEQ ID NO: 9] and5′-GCAGGTCTCCAAGGAAGTACG-3′ [SEQ ID NO: 11]) were used to amplifyportions of the SEQ ID NO:2 sequence from the samples. Amplifiedproducts were separated on an agarose gel, transferred and chemicallylinked to a nylon filter. The filter was then hybridized with aradioactively labeled (α³³P-dCTP) double-stranded probe generated fromthe full-length SEQ ID NO:2 sequence using a Klenow polymerase,random-prime method. The filters were washed (high stringency) and usedto expose a phosphorimaging screen for several hours. Bands indicatedthe presence of cDNA including SEQ ID NO:2 sequences in a specificlibrary, and thus mRNA expression in the corresponding cell type ortissue.

[0240] Of the 18 human tissues tested, macrophage was the only samplethat provided a signal, indicating that expression of SEQ ID NO:2 istightly regulated. In contrast, the CD39 molecule has been found intissues such as placenta, lung, skeletal muscle, kidney and heart.

EXAMPLE 4 Chromosomal Localization of the Gene Corresponding to SEQ IDNOs:1 and 2

[0241] Chromosome mapping technologies allow investigators to link genesto specific regions of chromosomes. Assignment to chromosome 14 wasperformed with the Coriell cell repository monochromosomal panel #2(NIGMS cell repository). This human rodent somatic cell hybrid panelconsists of DNA isolated from 24 hybrid cell cultures retaining 1 humanchromosome each. The panel was screened with gene-specific primers(5′-GCTACCTCACTTCCTTTGAG-3′ [SEQ ID NO: 9] and5′-CTGGCTGGTGAAGTTTTCCTC-3′ [SEQ ID NO: 10]) that generated a sequencetag site (STS). The Genebridge 4 radiation hybrid panel was alsoscreened (Research Genetics), and the results of the PCR screening weresubmitted to the Whitehead/MIT Radiation Hybrid mapping email server athttp://www-genome.wi.mit.edu.

EXAMPLE 5 Platelet Aggregation Assay

[0242] Blood is anticoagulated with 0.1 volume 3.2% sodium citrate.Platelet-rich plasma (PRP) is prepared with an initial whole bloodcentrifugation (200×g, 15 min., 25° C.) and a second centrifugation ofthe PRP (90×g, 10 min.) to eliminate residual erythrocytes andleukocytes. The stock suspension of PRP is maintained at roomtemperature under 5% CO₂-air. The platelet aggregation assay uses atwo-sample, four-channel Whole Blood Lumi-Aggregometor, model 560(Chronolog Corp., Havertown, Pa.). PRP containing 1.22×10⁸ platelets ispreincubated with the sample to be tested for inhibition of aggregationfor 10 min. at 37° C. in a siliconized glass cuvette containing astirring bar, followed by stimulation with either ADP (5 mm), collagen(5 mg/ml), or thrombin (0.1 unit/ml). Platelet aggregation is recordedfor at least 10 min. Data are expressed as the percentage of lighttransmission with platelet-poor plasma equal to 100%.

EXAMPLE 6 CD39-L4 is a Soluble Apyrase

[0243] The mammalian ectoapyrase CD39 is an integral membrane proteinwith two transmembrane domains (one at each end of the protein)(Maliszewski, C. R. et al., J. Immunol. 153:3574-3583). Thehydrophobicity profiles for the deduced amino acid sequence of otherfamily members, such as CD39L1 and CD39L3, are very similar to CD39(Chadwick, B. P. and Frischauf A. M., Genomics 50:357-367), suggestingthat these proteins also have two membrane spanning domains. However,CD39-L4 does not appear to have a second transmembrane domain at itsC-terminus, suggesting that the N-terminus hydrophobic region could codefor a secretory signal. To test this hypothesis, CD39-L4 was subclonedinto the mammalian expression vector pCDNA3.1 and a 6-Histidine tag wasinserted into the coding sequence.

[0244] The CD39-L4 cDNA sequence was initially isolated from amacrophage cDNA library (Invitrogen). The sense primer(5′-TTAAAGCTTGGGAAAAGAATGGCCACTTC-3′, SEQ ID NO. 20) with a HindIII siteand the antisense primer (5′-AGACTCGAGGTGGCTCAATGGGAGATGCC-3′, SEQ IDNO. 21) with a XhoI site were used to subclone the coding sequences intothe mammalian expression vector pcDNA3.1 (Invitrogen). The nucleotidesequence of the insert is set forth in SEQ ID NO. 4. In order toimmunologically detect the protein, the coding region was furthermodified so that it would include a Gly-Ser6His epitope tag immediatelyfollowing Arg²⁴. Briefly, two partially overlapping complementaryoligonucleotides (5′-GCGCTGTCTCCCACAGAGGATCGCATCACCATCACCATCACAACCAGCAGACTTGGTT-3′ (SEQ ID. NO. 22) and5′-AACCAAGTCTGCTGGTTGTGATGGTGATGGTGATGCGATCCTCTGTGGG AGACAGCGC-3′ (SEQID NO. 23)) were used on the CD39-L4 pcDNA3.1 template. The primers wereextended in opposite directions around the plasmid using a 12 cycle PCRprogram (95° C., 1 minute; 60° C., 1 minute; 72° C., 15 minutes)(Stratagene). The reaction was treated with DpnI to digest themethylated parental DNA and then transformed into E. coli. Colonies werescreened for the insert.

[0245] To ascertain whether CD39-L4-6His is secreted, the coding regionof the CD39-L4-6His protein was inserted into the pcDNA3.1 expressionvector and transiently transfected into COS-7 cells. Cos-7 cellsobtained from the American Tissue Type Culture Collection were grown inDMEM supplemented with 10% FBS and 100 units/ml penicillin G and 100μg/ml streptomycin sulfate at 37° C. in 10% CO₂. Transfections wereperformed at 75% confluency in 10 cm plates with Fugene-6 according tothe manufacturers instructions. The cells in 7 mls of medium wereincubated with 16 μl of Fugene-6 and 8 μg of DNA for 14-18 hours. At theend of the transfection the medium was replaced with DMEM mediumcontaining low serum (1% FBS). The cells were then incubated for 24-48hours prior to harvesting.

[0246] The CD39-L4-6His was concentrated by treating the cell lysatesand medium with Nickel-NTA agarose (Qiagen) followed by SDS/PAGE andimmunoblot analysis with an antibody against the Arg-Gly-Ser-6Hisepitope. Cells were washed twice with PBS containing 0.5 μg/mlleupeptin, 0.7 μg/ml pepstatin and 0.2 μg/ml aprotinin. After a briefsonication and centrifugation step to clear the lysate, the samples werethen incubated with a Nickel-NTA resin at 4° C. for 2-3 hours. Thehistidine-tagged protein complexed to the resin was washed three timeswith PBS before loading onto a 10% SDS/PAGE gel for Western blotanalysis. CD39-L4 was detected in both the cell lysate and the mediumfrom cells transfected with the CD39-L4-6His expression vector, but notfrom control cells. While the predicted molecular weight of CD39-L4-6Hisis 46 kDa, the immunoreactive protein exhibited a mobility by SDS/PAGEcorresponding to a molecular mass of approximately 51 kDa in the mediaand approximately 48 kDa in the cell lysate. The difference in apparentmolecular weight may be due to posttranslational medications of threepotential N-glycosylation sites in the CD39-L4 predicted amino acidsequence.

[0247] Secretion of CD39-L4 was also examined by treatment of thetransfected cells with brefeldin A, an inhibitor of translocation ofsecretory proteins from the endoplasmic reticulum to the Golgiapparatus. Chadwick, et al., Genomics 50:357-367 (1998). Brefeldin A wasdissolved in ethanol and added to the transfected cells 48 hours aftertransfection. Both control and brefeldin A treated cells were washedonce with PBS and incubated for 8 hours in medium with none or varyingdosages of brefeldin A. Increasing dosages of brefeldin A blockedsecretion of CD39-L4-6His and led to massive intracellular accumulation.

EXAMPLE 7 Assay For ATPase Activity

[0248] Apyrase activity was determined by measuring the amount of[³³P]P_(i) released from [γ³³P]ATP. In summary, 50 μl of samples wereincubated in the presence of 10 μCi of [γ³³P]ATP for one hour at 37° C.The [33P]P_(i) released and the [γ³³P]ATP were separated by thin layerchromatography (TLC) plates (EM Science). The solvent system consistedof 1 M KH₂PO₄. The separated compounds were scanned for radioactivitywith a Phosphoimager (Molecular Dynamics, Sunnyvale, Calif.) andquantitated by ImageQuant software. COS-7 cells transfected with SEQ IDNOs. 3 and 25 had at least a four fold increase in activity over cellstransfected with the vector alone. Although ATPase activity was present,Example 13 demonstrates that CD39-L4 has significantly more NDPaseactivity.

EXAMPLE 8 Site-directed Mutagenesis of CD39L4

[0249] Site directed mutagenesis was employed to increase the enzymaticactivity of CD39L4. Amino acid sequence comparisons between CD39 familymembers reveal four highly homologous regions in all five human members(Chadwick and Frischauf, Genomics 50:357-367, 1998). These regions,termed apyrase-conserved regions (ACRs), are present not only in theCD39 family members but other apyrases from species as distant as yeastand plants. Examination of similarities and differences in the CD39 ACRsled to the design of three CD39L4 mutants (see FIG. 5). In thesemutants, codons encoding CD39 ACR specific residues were used to replacecodons from the CD39L4 wild type ACR sequence. Only residues withsignificantly different structural or chemical properties were replaced.A PCR based approach was used to produce these mutations.

[0250] Briefly, the expression vector pCDNA3.1 (Invitrogen) containingthe full coding sequence of the CD39L4 gene (with a 6 Histidine taginserted after Arg 24 in the coding sequence to allow purification ofthe secreted mature form of the protein) was subjected to a PCR-basedsite-directed mutagenesis approach using overlapping oligonucleotides[CD39-L4 ACR I mutant (nt 177-148 and 160-204): 5′-GTG AGT GCT CCC TGCATC TAA CAT AAT TCC-3′ (SEQ ID NO: 12) and 5′-GAT GCA GGG AGC ACT CACACT AGT ATT CAT GTT TAC ACC TTT GTG-3′ (SEQ ID NO: 13); CD39-L4 ACR IImutant (nt 402-359 and 385-415): 5′-GCG TAG TCC TGC TGT TGC CCC TAG GTACAC TGG GGT CTT TTA CC-3′ (SEQ ID NO: 14) and 5′-GCA ACA GCA GGA CTA CGCTTA CTG CCA GAA C-3′ (SEQ ID NO: 15); and CD39-L4 ACR III mutant (nt532-485 and 513-540): 5′-CCC AAG CGA ATA TGC CTT CGT CTT GTC CAG TCA TGATGC TAA CAC TGC-3′ (SEQ ID NO: 16) and 5′-CGA AGG CAT ATT CGC TTG GGTTAC TGT G-3′ (SEQ ID NO:17)]. After amplification of the whole plasmidwith Pfu DNA polymerase (Stratagene) (95° C./1 min; 60° C./1 min; 72°C./15 min for 12 cycles), the methylated parental DNA was digested withthe restriction enzyme DpnI, leaving only the unmethylated PCR amplifiedproducts. The resulting annealed double-stranded nicked products werethen transformed into bacteria and the resulting colonies were screenedfor the desired mutations by sequencing. The subsequent constructs werefully sequenced to verify that the mutations were in fact introduced andthat no extraneous mutations were generated.

EXAMPLE 9 ACR III Mutant Increases ADPase Activity

[0251] Plasmids containing the mutated and wild type forms of the CD39L4gene were transfected into COS-7 cells. After two days, protein waspurified from the culture medium using a Nickel-NTA resin approach toconcentrate the tagged proteins. These proteins were then assayed forATPase and ADPase activity by measuring the inorganic phosphate released(Wang, T. F., et al., J. Biol. Chem. 273:24814-24821, 1998). Theproteins were incubated in apyrase buffer (15 mM Tris pH 7.4, 135 mMNaCl, 2 mM EGTA and 10 mM glucose) for 1 hour at 37° C. with or without2 mM CaCl₂ or 2 mM MgCl₂. Phosphatase reactions were initiated by theaddition of ADP or ATP to a final concentration of 1 mM. The reaction ofinorganic phosphorus with ammonium molybdate in the presence of sulfuricacid, produces an unreduced phosphomolybdate complex. The absorbance ofthis complex at 340 nm is directly proportional to the inorganicphosphorus concentration (Daly, J. A., and Ertingshausen G., Clin. Chem.18:263 (1972) (Sigma Diagnostics)).

[0252] As seen in FIG. 7, mutations in ACR I and II eliminate activity,whereas the mutations in ACR III increase activity six-fold over wildtype. This increased activity therefore offers a greater therapeuticpotential, as less protein could be administered to offer the samepharmacological effect. The replacement of three amino acids in the IIIregion (amino acids 167 to 181 in CD39-L4) and the resulting increase inADPase activity predicts that replacement of additional amino acidswithin this region by amino acids from the equivalent region of CD39 mayalso enhance the activity of the protein over wild type CD39L4. Theincrease in ADPase activity over wild type may also be due to thereplacement of only one or two of the three amino acids; this can beconfirmed by replacing one or two amino acids at a time.

[0253] The polynucleotide and amino acid sequences of a CD39-L4 varianttermed ACRIII and having the amino acid substitutions D168-T, S170-Q andL175-F compared to wild type CD39-L4 (SEQ ID NO: 5) are set forth in SEQID NOs: 6 and 7, respectively, and in FIG. 6.

EXAMPLE 10 ACR III Mutant and Wild Type Forms Are Specific For ADP andNot ATP

[0254] Both the CD39L4 wild type and the CD39L4 variant with mutationsin the ACRIII region hydrolyze ADP. However, when ATP was tested as asubstrate, neither the CD39L4 nor the CD39L4 mutant, ACR III, catalyzedhydrolysis. In contrast, CD39 as a membrane bound molecule (Marcus, etal., The Journal of Clinical Investigation, 99: 1351-1360) or as agenetically engineered soluble form (Gayle, et al., The Journal ofClinical Investigation, 101:1851-1858, 1998) is able to hydrolyze bothATP and ADP substrates efficiently. The specificity that both CD39L4wild type and the CD39L4 ACR III mutant have for ADP is an advantageousfeature that makes these CD39L4-type molecules better antiplatelettherapeutic candidates than CD39, as ADP is the agonist that causesplatelet aggregation. Therapeutics that have both ADPase and ATPaseactivities potentially could create adverse side effects by interferingwith levels of ATP in the circulation.

EXAMPLE 11 Organization of the Human CD39-L4 Gene

[0255] A human CITB BAC genomic library (Research Genetics) was screenedwith gene specific primers [246-16 (nt 5522-5543),5′-CTTCCTTCACTGGGAATTCAGG-3′ (SEQ ID NO: 18) and 246-K4 (nt 4922-4945),5′-CTGTTTACCGAGATGGTTGGAAGC-3′ (SEQ ID NO: 19)] using a PCR based assay.

[0256] Briefly, gene specific primers were used to screen pools of BACDNAs. BAC pools that produced an amplified DNA fragment of the predictedsize were pursued until an individual BAC was identified. BAC63-I18 wasisolated and sequenced with gene specific primers for the CD39-L4 cDNA,as well as intron specific primers. The CD39-L4 coding sequence wasfound to be distributed over 10 exons spanning 9.3 kb of genomic DNA asset out in SEQ ID NO: 8.

EXAMPLE 12 CD39-L4 and CD39-L2 Are Stimulated By Divalent Cations

[0257] The high degree of conservation in the apyrase conserved regionsof CD39-L4 and CD39-L2 suggests similar function to other apyrases. Totest this hypothesis, COS-7 cells were transfected with the CD39-L4-6Hisand CD39-L2myc-His construct as described herein. The medium fromtransfected cells was incubated with Nickel-NTA resin (Qiagen) in orderto capture the 6His tagged protein, the resin was washed with assaybuffer (buffer A, 15 mM Tris pH 7.5, 134 mM NaCl and 5 mM glucose) andthe protein still tethered to the resin in a suspension was assayed forADPase activity. Nucleotidase activity was determined by measuring theamount of inorganic phosphate released from nucleotide substrates usingthe technique of Dlay and Ertingshausen, Clin. Chem. 18:263-265 (1972).In this reaction the complex of inorganic phosphorus with phosphorreagent (ammonium molybdate in the presence of sulfuric acid) producesan unreduced phosphomolybdate compound. The absorbance of this complexat 340 nm is directly proportional to the inorganic phosphorusconcentration. The protein still tethered to the resin as a 30% (50% forCD39-L2) suspension in buffer A was assayed by the addition of thenucleotide to a final concentration of 1 mM and incubated at 37° C. for30 minutes. The reaction was stopped by adding 100 volumes of phosphorreagent. The amount of phosphate released from the reaction wasquantified using a calcium/phosphorus combined standard (Sigma). Theamount of protein used in the assays was estimated by comparing theintensity of the bands in Western blots with that of a series ofstandards of known quantity. CD39-L4 protein from transfected cellsdisplayed a 2.3 fold increase in activity over the cells transfectedwith the vector alone. When Ca²⁺ and Mg²⁺ were added, the activityincreased 3.6 fold and 6 fold, respectively. CD39-L2 protein fromtransfected cells displayed an 8.7 fold increase in activity over thecells transfected with the vector alone. When Ca²⁺ and Mg²⁺ were added,the activity of the CD39-L2 cells increased another 2-3 fold.

EXAMPLE 13 Characterization of CD39-L4 Activity

[0258] CD39-L4 protein was assayed for ADPase activity in the presenceof different kinds of inhibitors of ADPases. Control ecto-apyraseactivity was determined with protein tethered to the Nickel-NTA resin.Both assays were performed as described above except the protein was inbuffer A containing 2 mM CaCl₂ and 2 mM MgCl₂. As shown by Table 1below, inhibitors of phosphatases (F⁻) and adenylate kinase (Ap5A) didnot inhibit activity. The inhibitors of vacuolar ATPases (NEM),mitochondrial ATPases (N3⁻) and Na⁺, K⁺, ATPase (ouabain) did notsignificantly inhibit the Ca²⁺ and Mg²⁺ stimulated activity. However,metal chelators (EDTA and EGTA) significantly inhibited activity. Theseresults show that the overwhelming majority of the activity in theassays originates from a protein bound to the resin with characteristicsof an E-type apyrase. TABLE 1 Inhibition of CD39-L4 activity INHIBITORS% OF CONTROL Control 100 ± 7 Ouabain (1 mM)  96 ± 6 NEM (10 mM) 106 ± 5N3⁻ (1 mM)  100 ± 12 F⁻ (10 mM) 113 ± 5 Ap5A (10 μM) 121 ± 9 EGTA (2 mM) 35 ± 3 EDTA (2 mM)  52 ± 3

[0259] As shown in Table 2 below, the nucleotide specificity of CD39-L4was also assayed as described above. The CD39-L4 activity was determinedwith protein tethered to the Ni-NTA resin. The protein was in buffer Acontaining 1 mM EGTA, as well as 2 mM CaCl₂ and MgCl₂. The assay wasstarted by adding the nucleotides to a final concentration of 1 mM. Thevalues below are expressed relative to ADP. The relative activity of thenucleotide triphosphates varies almost seven-fold with ATP being thepoorest substrate. No phosphate release was detected with AMP and ADPwas hydrolyzed at a rate approximately twenty-fold higher than ATP. Theother nucleotide diphosphates (GDP and UDP) were also very efficientlyhydrolyzed by CD39-L4. These results indicate that CD39-L4 defines a newclass of E-type apyrase in humans with a specificity for NDPs asenzymatic substrates. TABLE 2 Substrate specificity of CD39-L4NUCLEOTIDE % OF CONTROL ADP 100 ± 15  ATP 5 ± 1 AMP 0 CTP 26 ± 2  GTP 34± 1  UTP 12 ± 4  CDP 268 ± 11  GDP 334 ± 38  UDP 408 ± 14 

EXAMPLE 14 Glycosylation Is Not Essential For CD39-L4 Activity

[0260] Posttranslational modifications such as N-linked glycosylationare common in secreted and membrane-bound mammalian proteins. Thesemodifications may be important for correct protein folding or enzymaticactivity and are not easily reproduced when the proteins are expressedin other organisms such as bacteria. In order to test whether CD39-L4 isglycosylated, COS-7 cells, transfected as described in Example 6, weretreated with tunicamycin (Sigma), which blocks the formation ofN-glycosidic linkages.

[0261] COS-7 cells were grown to 75% confluency and transfected with theCD39-L4-6His construct. After 24 hours, a fraction of the COS-7 cellswere treated with Tunicamycin at a concentration of 5 μg/ml. The mediawas replaced again after 24 hours with fresh tunicamycin and harvestedafter 48 hours. The CD39-L4-6His protein was concentrated by treatingthe media with Nickel-NTA agarose (Qiagen). The resin was washed withassay buffer and the protein still tethered to the resin in a suspensionwas assayed for a shift in electrophoretic mobility as well as itsADPase activity.

[0262] Western blot analysis using an antibody against the 6-His epitoperevealed that the glycosylated CD39-L4 protein isolated from the controlcells had an approximate size of 51 kDa. However, tunicamycin treatedcells had a molecular weight of approximately 46 kDa indicating that theprotein was deglycosylated.

[0263] ADPase activity of the tunicamycin treated cells was assayed asdescribed in Example 12 above. The deglycosylated CD39-L4 protein hadADPase activity comparable to an equal amount of the glycosylatedprotein isolated from control cells. This demonstrates thatglycosylation of the protein is not important for ADPase activity.

EXAMPLE 15 Cloning and Expression of CD39-L2

[0264] The CD39-L2 coding sequence (SEQ ID NO: 26) was subcloned intopcDNA3.1/myc-His(+)A (Invitrogen) via the EcoRI and XbaI sites. Briefly,a human adult heart cDNA library (Gibco BRL) was subjected to polymerasechain reaction (PCR) using gene-specific primers L2-5′B(5′-CGTATCCCGCGGGTGGAGGCCGGGGTG-3′, SEQ ID NO: 28) and L2-3′B(5′-CTTCTGCAAGTCCCAGAGCCAGTGTGC-3′, SEQ ID NO: 29). The resultingproducts were diluted 100-fold and subjected to a second round of PCRwith primers L2-5′A (5′-GGAGCCCAAAAGACCGGCTGC-3′, SEQ ID NO: 30) andL2-3′A (5′-TGAAGTCACGTCCAGGACAGG-3′, SEQ ID NO: 31). The productrepresented a single band by agarose gel and was purified and sequencedto confirm its identity. Primers corresponding to the translationalstart region and the carboxy terminal region, excluding the stop codon,of the CD39-L2 coding sequence, L2EcoMet(5′-CGGAATTCAACATGAAAAAAGGTAATCCGTTATGAA-3′, SEQ ID NO: 32) and L2Xba3′(5′-TGTCTAGATGAGGCTGGACTCTTCTG-3′, SEQ ID NO: 33) were used on thepurified DNA to produce a DNA fragment corresponding to the entirecoding region of the CD39-L2 gene, flanked by EcoRI and XbaI sites. ThisPCR product was digested to generate overhang ends that were ligatedinto the EcoRI and XbaI sites of pcDNA3.1/myc-His(+)A. The resultingplasmid allowed expression of the CD39-L2 coding sequence fused in framewith the myc-6His epitope at the carboxy terminus.

[0265] Transfection of COS-7 cells was performed as described below.COS-7 cells obtained from the American Tissue Type Culture Collectionwere grown in MDEM supplemented with 10% FBS and 100 units/ml penicillinG and 100 μg/ml streptomycin sulfact at 37° C. in 10% CO₂. Transfectionswere performed at 75% confluency in 10 cm plates with Fugene-6 accordingto the manufacturer's instructions. The cells in 10 ml of medium wereincubated with 16 μl of Fugene-6 and 8 μg of DNA for 48 hours. Themedium was then replaced by DMEM containing low serum (1% FBS), andincubated for 48 hours before harvesting.

EXAMPLE 16 Cellular Localization of CD39-L2

[0266] Western blot analysis was performed on COS cells transfected withthe plasmid described in Example 15 above to determine the cellularlocalization of CD39-L2. To detect myc epitope tagged recombinantproteins, the blot was incubated with a 2000-fold dilution of theanti-c-myc monoclonal antibody (Invitrogen) at room termperature for twohours. The secondary antibody (anti-mouse Ig AP conjugate) was diluted1000-fold and incubated for 1-2 hours at room temperature. Boundantibody was detected by using Sigma Fast™ 5-bromo4-chloro-3indolylphosphate/nitro blue tetrazolium (BCIP/NTP) as the alkaline phosphatasesubstrate according to instructions of the manufacturer.

[0267] The recombinant protein was detected in the media and themembrane fractions of the CD39-L2 transfected cells, but not in thecytosolic fraction or control transfections. The relative bandintensities suggest that the majority of the recombinant CD39-L2 proteinis secreted into the media and a fraction resides in the membrane. Thepredicted molecular weight of unprocessed CD39-L2 is 53 kD. However, themembrane and secreted fractions displayed slower mobility by SDS/PAGEthan that predicted by its amino acid content, suggesting posttranslational modification.

[0268] To confirm that recombinant CD39-L2 is secreted, the cellularlocalization was performed using increasing amounts of brefeldin A, aninhibitor of translocation of secretory proteins from the endoplasmicreticulum to the Golgi apparatus. Recombinant CD39-L2 in the mediadecreased in a brefeldin A dose dependent manner. Recombinant CD39-L2also accumulated in the cytosol in a dose dependent manner. Therefore,recombinant CD39-L2 secretion follows the conventional cellularsecretory pathway.

[0269] Flow cytometric analysis was used to determine if recombinantCD39-L2 is expressed on cell surfaces. COS-7 cells were transfected asdescribed above with either pcDNA3.1/myc-His(+)A or pCD39-L2myc-HIS.After 72 hours of transfection, the cells were washed twice with PBS,and dislodged with 10 mM EDTA in PBS. Cells were pelleted bycentrifugation at 300 g for five minutes, washed with PBS andresuspended in binding buffer (PBS containing 3% FBS and 0.02% sodiumazide) at a concentration of 1×10⁶ cells per 100 μl. The cells werefirst stained with 20 μg/ml of monoclonal anti-myc antibody for 30minutes at 4° C. The cells were then washed with binding buffer andstained with 20 μg/ml of R-phycoerythrin conjugated goat anti-mouse IgGantibody (Molecular Probes, Eugene, Oreg.). After washing with bindingbuffer, the cells were resuspended in 1 ml of binding buffer andanalyzed on the FACScalibur flow cytometer (Becton DickinsonImmunocytometry Systems, San Jose, Calif.).

[0270] Expression of cell surface recombinant CD39-L2 was found only oncells transfected with pCD39-L2myc-His, while cells from the controltransfection showed no antibody binding. These results confirm thatCD39-L2 is a secreted apyrase.

EXAMPLE 17 Characterization of CD39-L2 Activity

[0271] CD39-L2 protein was assayed for ADPase activity in the presenceof different kinds of inhibitors of ADPases. Control ecto-apyraseactivity was determined with protein tethered to the Nickel-NTA resin.Both assays were performed as described in Example 12 above except theprotein was in buffer A containing 1 mM EGTA and 3 mM CaCl₂. The assaywas started by adding ADP to 1 mM followed by a 30 minute incubation at37° C. As shown by Table 3 below, CD39-L2 is not inhibited by inhibitorsof vacuolar adenosine triphospatase (ATPases) (NEM), mitochondrialATPase (N₃ ⁻) and Na⁺, K⁺ ATPase (oubain). An inhibitor of adenylatekinase (Ap5A) did not inhibit activity, while an inhibitor ofphosphatases (F⁻) partially inhibited activity. Metal chelators (EDTAand EGTA) inhibited CD39-L2 activity thereby demonstrating that CD39-L2activity is dependent on divalent cations. TABLE 3 Inhibition of CD39-L2activity INHIBITORS % OF CONTROL Control 100 ± 3  Ouabain (1 mM) 101 ±9  NEM (10 mM) 88.4 ± 13   N₃ ⁻ (1 mM) 90 ± 13 F⁻ (10 mM) 63 ± 9  Ap5A(10 μM) 87 ± 11 EGTA (2 mM) 34 ± 10 EDTA (2 mM) 18.4 ± 9  

[0272] As shown in Table 4 below, the nucleotide specificity of CD39-L2was also assayed as described in Example 12. The CD39-L2 activity wasdetermined with protein tethered to the Ni-NTA resin. The protein was inassay buffer A containing 1 mM EGTA, 3 mM CaCl₂ and 3 mM MgCl₂. Theassay was started by adding the nucleotides to a final concentration of1 mM. The values are expressed relative to ADP. The samples were assayedat 37° C. for 30 minutes. TABLE 4 Substrate Specificity of CD39-L2NUCLEOTIDE % OF CONTROL ADP 100 ± 8  ATP 16 ± 2 AMP 0.6 ± 1  CTP 44 ± 4GTP 39 ± 1 UTP 13 ± 1 CDP 282 ± 18 GDP 338 ± 52 UDP 303 ± 5 

[0273] These results confirm that CD39-L2 along with CD39-L4 define anew class of E-type apyrase in humans with a specificity for NDPs asenzymatic substrates.

EXAMPLE 18 CD39-L4 and CD39-L2 Expression Using In Situ Hybridization

[0274] A. In situ hybridization of CD39-L4 in kidney

[0275] A 298 nt fragment of the CD39L4 cDNA 3′-untranslated region wasamplified by PCR with oligonucleotide primers 246D13 and 246D4(5′-ATCCTGGACTTGAGCCTAGAG-3′, SEQ ID NO: 34 and5′-CTGATATTGATGGGTCTTGGG-3′, SEQ ID NO: 35). The fragment was subclonedinto the pCR™ II-TOPO plasmid (Invitrogen) and sense and antisense RNAwere synthesized. The probe was labeled using the digoxigenin labelingkit supplied by Boehringer-Mannheim as described in the manufacturersprotocol. Automated in situ hybridization was performed by QualTekMolecular Labs (Santa Barbara, Calif.) using a modified version of apreviously published procedure (Myers, J. A., et al., (1995) J. Surg.Path. 1, 191-203). The Ventana Medical Systems, Inc. (Tucson, Ariz.)TechMate™ Automated Staining System was used for this procedure. Alltissues were fixed in 10% neutral buffered formalin, paraffin-embeddedand cut into 4 μm thick sections. Sections were placed onto Ventana'sChemMate™ Capillary Gap Slides (POP075).

[0276] Staining of kidney sections revealed that specific cell typeshybridized with the antisense probe but not the sense probe in a highlyspecific manner. The staining of the glomerulus revealed that theepithelia of the Bowman's capsule, podocyte epithelia and mesangialcells were specifically stained. The expression of the CD39L4 protein inthis region could be necessary to prevent platelet aggregation in theBowman's capsule because platelets become highly concentrated in thisparticular region as water and ions are filtered from the blood. Thebloody region within the kidney showed staining of white blood cells,presumably macrophages. This staining is consistent with previousstudies where a macrophage cDNA library showed expression of the CD39L4cDNA. CD39L4 staining was also found in some tubule epithelial cells inthe kidney.

[0277] B. In situ hybridization of CD39-L2 in heart

[0278] A 186 nt fragment of the CD39L2 cDNA was amplified by PCR witholigonucleotide primers L2RNA3 and L2RNA2 (5′-GGATGGAAAGGAGTTGGTCAG-3′,SEQ ID NO: 36 and 5′-GTCCACATGCTTCACTTCCTC-3′ SEQ ID NO: 37). Thefragment was subcloned into the pCR™ II-TOPO plasmid (Invitrogen) andsense and antisense RNA were synthesized and labeled as described above.Automated in situ hybridization was performed as described above.

[0279] Staining of heart sections revealed that specific cell typeshybridized with the antisense probe but not the sense probe in a highlyspecific manner. The cardiac muscle cells as well as capillaryendothelial cells and white blood cells within a blood vessel showedspecific staining. This staining is consistent with previous studieswhere a heart cDNA library showed robust expression of the CD39L2 cDNA.

[0280] This in situ hybridization data is consistent with aphysiological role for CD39-L4 and CD39-L2 in regulating plateletaggregation and hemostasis. Further in situ hybridization may be carriedout to confirm this activity.

[0281] The present invention is not to be limited in scope by theexemplified embodiments which are intended as illustrations of singleaspects of the invention, and compositions and methods which arefunctionally equivalent are within the scope of the invention. Indeed,numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe present preferred embodiments. Consequently, the only limitationswhich should be placed upon the scope of the invention are those whichappear in the appended claims. All references cited within the body ofthe instant specification are hereby incorporated by reference in theirentirety.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 39 <210> SEQ ID NO 1<211> LENGTH: 300 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 ggcatattag cttgggttac tgtgaatttt ctgacaggtc agctgcatggccacagacag 60 gagactgtgg ggaccttgga cctaggggga gcctccaccc aaatcacgttcctgccccag 120 tttgagaaaa ctctggaaca aactcctagg ggctacctca cttcctttgagatgtttaac 180 agcacttata agctctatac acatagttac ctgggatttg gattgaaagctgcaagacta 240 gcaaccctgg gagccctgga gacagaaggg actgatgggc acactttccggagtgcctgt 300 <210> SEQ ID NO 2 <211> LENGTH: 1799 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (246)..(1529) <221> NAME/KEY: misc_feature <222> LOCATION:(1718) <223> OTHER INFORMATION: n = adenine or guanine or cytosine orthymine <400> SEQUENCE: 2 gcgggctgcc gcgcaagggt ggcgcgcgcg cgttttccttgttcctggtc aacaaagaaa 60 tgtggagtgt cttggctgaa tcctcataca gacaagatcattatggtgct gttaggttga 120 aaaagtgata taataaagga accaaggaga aaattcagaaggaaagaaaa aattgcctct 180 gcaggtgtgc gagcaggatt gcttctgcaa caaaagcctccacccagcca catcttggga 240 aaaga atg gcc act tct tgg ggc aca gtc ttt ttcatg ctg gtg gta tcc 290 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met LeuVal Val Ser 1 5 10 15 tgt gtt tgc agc gct gtc tcc cac agg aac cag cagact tgg ttt gag 338 Cys Val Cys Ser Ala Val Ser His Arg Asn Gln Gln ThrTrp Phe Glu 20 25 30 ggt atc ttc ctg tct tcc atg tgc ccc atc aat gtc agcgcc agc acc 386 Gly Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser AlaSer Thr 35 40 45 ttg tat gga att atg ttt gat gca ggg agc act gga act cgaatt cat 434 Leu Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg IleHis 50 55 60 gtt tac acc ttt gtg cag aaa atg cca gga cag ctt cca att ctagaa 482 Val Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu65 70 75 ggg gaa gtt ttt gat tct gtg aag cca gga ctt tct gct ttt gta gat530 Gly Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp 8085 90 95 caa cct aag cag ggt gct gag acc gtt caa ggg ctc tta gag gtg gcc578 Gln Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala 100105 110 aaa gac tca atc ccc cga agt cac tgg aaa aag acc cca gtg gtc cta626 Lys Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu 115120 125 aag gca aca gca gga cta cgc tta ctg cca gaa cac aaa gcc aag gct674 Lys Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys Ala 130135 140 ctg ctc ttt gag gta aag gag atc ttc agg aag tca cct ttc ctg gta722 Leu Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu Val 145150 155 cca aag ggc agt gtt agc atc atg gat gga tcc gac gaa ggc ata tta770 Pro Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu 160165 170 175 gct tgg gtt act gtg aat ttt ctg aca ggt cag ctg cat ggc cacaga 818 Ala Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg180 185 190 cag gag act gtg ggg acc ttg gac cta ggg gga gcc tcc acc caaatc 866 Gln Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile195 200 205 acg ttc ctg ccc cag ttt gag aaa act ctg gaa caa act cct aggggc 914 Thr Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly210 215 220 tac ctc act tcc ttt gag atg ttt aac agc act tat aag ctc tataca 962 Tyr Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr225 230 235 cat agt tac ctg gga ttt gga ttg aaa gct gca aga cta gca accctg 1010 His Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu240 245 250 255 gga gcc ctg gag aca gaa ggg act gat ggg cac act ttc cggagt gcc 1058 Gly Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe Arg SerAla 260 265 270 tgt tta ccg aga tgg ttg gaa gca gag tgg atc ttt ggg ggtgtg aaa 1106 Cys Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly ValLys 275 280 285 tac cag tat ggt ggc aac caa gaa ggg gag gtg ggc ttt gagccc tgc 1154 Tyr Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly Phe Glu ProCys 290 295 300 tat gcc gaa gtg ctg agg gtg gta cga gga aaa ctt cac cagcca gag 1202 Tyr Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu His Gln ProGlu 305 310 315 gag gtc cag aga ggt tcc ttc tat gct ttc tct tac tat tatgac cga 1250 Glu Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr AspArg 320 325 330 335 gct gtt gac aca gac atg att gat tat gaa aag ggg ggtatt tta aaa 1298 Ala Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly IleLeu Lys 340 345 350 gtt gaa gat ttt gaa aga aaa gcc agg gaa gtg tgt gataac ttg gaa 1346 Val Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp AsnLeu Glu 355 360 365 aac ttc acc tca ggc agt cct ttc ctg tgc atg gat ctcagc tac atc 1394 Asn Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu SerTyr Ile 370 375 380 aca gcc ctg tta aag gat ggc ttt ggc ttt gca gac agcaca gtc tta 1442 Thr Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser ThrVal Leu 385 390 395 cag ctc aca aag aaa gtg aac aac ata gag acg ggc tgggcc ttg ggg 1490 Gln Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp AlaLeu Gly 400 405 410 415 gcc acc ttt cac ctg ttg cag tct ctg ggc atc tcccat tgaggccacg 1539 Ala Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His420 425 tacttccttg gagacctgca tttgccaaca cctttttaag gggaggagagagcacttagt 1599 ttctgaacta gtctggggac atcctggact tgagcctaga gattwrgttaattaascggc 1659 cgagcttatc cttwatragg taatttactt gcmtggccgc gtttacacgtcgtgatggna 1719 aacctgcgtc ccaactaacg cttgasamat ccccttcgca gctgcgataccaaaagccga 1779 cgacgccttc cacagtgcca 1799 <210> SEQ ID NO 3 <211>LENGTH: 428 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:3 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 1015 Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 2530 Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 4045 Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 5560 Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 7075 80 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 8590 95 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys100 105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val LeuLys 115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala LysAla Leu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro PheLeu Val Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser AspGlu Gly Ile Leu Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly GlnLeu His Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu GlyGly Ala Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys ThrLeu Glu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met PheAsn Ser Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly PheGly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu ThrGlu Gly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro ArgTrp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln TyrGly Gly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 AlaGlu Val Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315320 Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325330 335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val340 345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu GluAsn 355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser TyrIle Thr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser ThrVal Leu Gln 385 390 395 400 Leu Thr Lys Lys Val Asn Asn Ile Glu Thr GlyTrp Ala Leu Gly Ala 405 410 415 Thr Phe His Leu Leu Gln Ser Leu Gly IleSer His 420 425 <210> SEQ ID NO 4 <211> LENGTH: 1287 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1284) <400> SEQUENCE: 4 atg gcc act tct tgg ggc aca gtcttt ttc atg ctg gtg gta tcc tgt 48 Met Ala Thr Ser Trp Gly Thr Val PhePhe Met Leu Val Val Ser Cys 1 5 10 15 gtt tgc agc gct gtc tcc cac aggaac cag cag act tgg ttt gag ggt 96 Val Cys Ser Ala Val Ser His Arg AsnGln Gln Thr Trp Phe Glu Gly 20 25 30 atc ttc ctg tct tcc atg tgc ccc atcaat gtc agc gcc agc acc ttg 144 Ile Phe Leu Ser Ser Met Cys Pro Ile AsnVal Ser Ala Ser Thr Leu 35 40 45 tat gga att atg ttt gat gca ggg agc actgga act cga att cat gtt 192 Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr GlyThr Arg Ile His Val 50 55 60 tac acc ttt gtg cag aaa atg cca gga cag cttcca att cta gaa ggg 240 Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu ProIle Leu Glu Gly 65 70 75 80 gaa gtt ttt gat tct gtg aag cca gga ctt tctgct ttt gta gat caa 288 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser AlaPhe Val Asp Gln 85 90 95 cct aag cag ggt gct gag acc gtt caa ggg ctc ttagag gtg gcc aaa 336 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu GluVal Ala Lys 100 105 110 gac tca atc ccc cga agt cac tgg aaa aag acc ccagtg gtc cta aag 384 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro ValVal Leu Lys 115 120 125 gca aca gca gga cta cgc tta ctg cca gaa cac aaagcc aag gct ctg 432 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys AlaLys Ala Leu 130 135 140 ctc ttt gag gta aag gag atc ttc agg aag tca cctttc ctg gta cca 480 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro PheLeu Val Pro 145 150 155 160 aag ggc agt gtt agc atc atg gat gga tcc gacgaa ggc ata tta gct 528 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp GluGly Ile Leu Ala 165 170 175 tgg gtt act gtg aat ttt ctg aca ggt cag ctgcat ggc cac aga cag 576 Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu HisGly His Arg Gln 180 185 190 gag act gtg ggg acc ttg gac cta ggg gga gcctcc acc caa atc acg 624 Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala SerThr Gln Ile Thr 195 200 205 ttc ctg ccc cag ttt gag aaa act ctg gaa caaact cct agg ggc tac 672 Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln ThrPro Arg Gly Tyr 210 215 220 ctc act tcc ttt gag atg ttt aac agc act tataag ctc tat aca cat 720 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr LysLeu Tyr Thr His 225 230 235 240 agt tac ctg gga ttt gga ttg aaa gct gcaaga cta gca acc ctg gga 768 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala ArgLeu Ala Thr Leu Gly 245 250 255 gcc ctg gag aca gaa ggg act gat ggg cacact ttc cgg agt gcc tgt 816 Ala Leu Glu Thr Glu Gly Thr Asp Gly His ThrPhe Arg Ser Ala Cys 260 265 270 tta ccg aga tgg ttg gaa gca gag tgg atcttt ggg ggt gtg aaa tac 864 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile PheGly Gly Val Lys Tyr 275 280 285 cag tat ggt ggc aac caa gaa ggg gag gtgggc ttt gag ccc tgc tat 912 Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val GlyPhe Glu Pro Cys Tyr 290 295 300 gcc gaa gtg ctg agg gtg gta cga gga aaactt cac cag cca gag gag 960 Ala Glu Val Leu Arg Val Val Arg Gly Lys LeuHis Gln Pro Glu Glu 305 310 315 320 gtc cag aga ggt tcc ttc tat gct ttctct tac tat tat gac cga gct 1008 Val Gln Arg Gly Ser Phe Tyr Ala Phe SerTyr Tyr Tyr Asp Arg Ala 325 330 335 gtt gac aca gac atg att gat tat gaaaag ggg ggt att tta aaa gtt 1056 Val Asp Thr Asp Met Ile Asp Tyr Glu LysGly Gly Ile Leu Lys Val 340 345 350 gaa gat ttt gaa aga aaa gcc agg gaagtg tgt gat aac ttg gaa aac 1104 Glu Asp Phe Glu Arg Lys Ala Arg Glu ValCys Asp Asn Leu Glu Asn 355 360 365 ttc acc tca ggc agt cct ttc ctg tgcatg gat ctc agc tac atc aca 1152 Phe Thr Ser Gly Ser Pro Phe Leu Cys MetAsp Leu Ser Tyr Ile Thr 370 375 380 gcc ctg tta aag gat ggc ttt ggc tttgca gac agc aca gtc tta cag 1200 Ala Leu Leu Lys Asp Gly Phe Gly Phe AlaAsp Ser Thr Val Leu Gln 385 390 395 400 ctc aca aag aaa gtg aac aac atagag acg ggc tgg gcc ttg ggg gcc 1248 Leu Thr Lys Lys Val Asn Asn Ile GluThr Gly Trp Ala Leu Gly Ala 405 410 415 acc ttt cac ctg ttg cag tct ctgggc atc tcc cat tga 1287 Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His420 425 <210> SEQ ID NO 5 <211> LENGTH: 428 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 5 Met Ala Thr Ser Trp Gly Thr ValPhe Phe Met Leu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser HisArg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met CysPro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 Tyr Gly Ile Met Phe Asp AlaGly Ser Thr Gly Thr Arg Ile His Val 50 55 60 Tyr Thr Phe Val Gln Lys MetPro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 75 80 Glu Val Phe Asp Ser ValLys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 90 95 Pro Lys Gln Gly Ala GluThr Val Gln Gly Leu Leu Glu Val Ala Lys 100 105 110 Asp Ser Ile Pro ArgSer His Trp Lys Lys Thr Pro Val Val Leu Lys 115 120 125 Ala Thr Ala GlyLeu Arg Leu Leu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 Leu Phe GluVal Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 LysGly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu Ala 165 170 175Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185190 Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195200 205 Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr210 215 220 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr ThrHis 225 230 235 240 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu AlaThr Leu Gly 245 250 255 Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr PheArg Ser Ala Cys 260 265 270 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile PheGly Gly Val Lys Tyr 275 280 285 Gln Tyr Gly Gly Asn Gln Glu Gly Glu ValGly Phe Glu Pro Cys Tyr 290 295 300 Ala Glu Val Leu Arg Val Val Arg GlyLys Leu His Gln Pro Glu Glu 305 310 315 320 Val Gln Arg Gly Ser Phe TyrAla Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330 335 Val Asp Thr Asp Met IleAsp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340 345 350 Glu Asp Phe Glu ArgLys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 Phe Thr Ser GlySer Pro Phe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 Ala Leu LeuLys Asp Gly Phe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400 LeuThr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His 420 425 <210> SEQ ID NO6 <211> LENGTH: 1287 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1284) <400> SEQUENCE:6 atg gcc act tct tgg ggc aca gtc ttt ttc atg ctg gtg gta tcc tgt 48 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15gtt tgc agc gct gtc tcc cac agg aac cag cag act tgg ttt gag ggt 96 ValCys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 atcttc ctg tct tcc atg tgc ccc atc aat gtc agc gcc agc acc ttg 144 Ile PheLeu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 tat ggaatt atg ttt gat gca ggg agc act gga act cga att cat gtt 192 Tyr Gly IleMet Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60 tac acc tttgtg cag aaa atg cca gga cag ctt cca att cta gaa ggg 240 Tyr Thr Phe ValGln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 75 80 gaa gtt tttgat tct gtg aag cca gga ctt tct gct ttt gta gat caa 288 Glu Val Phe AspSer Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 90 95 cct aag cag ggtgct gag acc gtt caa ggg ctc tta gag gtg gcc aaa 336 Pro Lys Gln Gly AlaGlu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100 105 110 gac tca atc ccccga agt cac tgg aaa aag acc cca gtg gtc cta aag 384 Asp Ser Ile Pro ArgSer His Trp Lys Lys Thr Pro Val Val Leu Lys 115 120 125 gca aca gca ggacta cgc tta ctg cca gaa cac aaa gcc aag gct ctg 432 Ala Thr Ala Gly LeuArg Leu Leu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 ctc ttt gag gtaaag gag atc ttc agg aag tca cct ttc ctg gta cca 480 Leu Phe Glu Val LysGlu Ile Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 aag ggc agtgtt agc atc atg act gga caa gac gaa ggc ata ttc gct 528 Lys Gly Ser ValSer Ile Met Thr Gly Gln Asp Glu Gly Ile Phe Ala 165 170 175 tgg gtt actgtg aat ttt ctg aca ggt cag ctg cat ggc cac aga cag 576 Trp Val Thr ValAsn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185 190 gag act gtgggg acc ttg gac cta ggg gga gcc tcc acc caa atc acg 624 Glu Thr Val GlyThr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195 200 205 ttc ctg ccccag ttt gag aaa act ctg gaa caa act cct agg ggc tac 672 Phe Leu Pro GlnPhe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr 210 215 220 ctc act tccttt gag atg ttt aac agc act tat aag ctc tat aca cat 720 Leu Thr Ser PheGlu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 agt tacctg gga ttt gga ttg aaa gct gca aga cta gca acc ctg gga 768 Ser Tyr LeuGly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 gcc ctggag aca gaa ggg act gat ggg cac act ttc cgg agt gcc tgt 816 Ala Leu GluThr Glu Gly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 tta ccgaga tgg ttg gaa gca gag tgg atc ttt ggg ggt gtg aaa tac 864 Leu Pro ArgTrp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 cag tatggt ggc aac caa gaa ggg gag gtg ggc ttt gag ccc tgc tat 912 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 gcc gaagtg ctg agg gtg gta cga gga aaa ctt cac cag cca gag gag 960 Ala Glu ValLeu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320 gtccag aga ggt tcc ttc tat gct ttc tct tac tat tat gac cga gct 1008 Val GlnArg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330 335 gttgac aca gac atg att gat tat gaa aag ggg ggt att tta aaa gtt 1056 Val AspThr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340 345 350 gaagat ttt gaa aga aaa gcc agg gaa gtg tgt gat aac ttg gaa aac 1104 Glu AspPhe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 ttcacc tca ggc agt cct ttc ctg tgc atg gat ctc agc tac atc aca 1152 Phe ThrSer Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 gccctg tta aag gat ggc ttt ggc ttt gca gac agc aca gtc tta cag 1200 Ala LeuLeu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400ctc aca aag aaa gtg aac aac ata gag acg ggc tgg gcc ttg ggg gcc 1248 LeuThr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415acc ttt cac ctg ttg cag tct ctg ggc atc tcc cat tga 1287 Thr Phe His LeuLeu Gln Ser Leu Gly Ile Ser His 420 425 <210> SEQ ID NO 7 <211> LENGTH:428 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 7580 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 9095 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys AlaLeu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe LeuVal Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Thr Gly Gln Asp GluGly Ile Phe Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln LeuHis Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly GlyAla Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr LeuGlu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe AsnSer Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe GlyLeu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr GluGly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg TrpLeu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala GluVal Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr IleThr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr ValLeu Gln 385 390 395 400 Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly TrpAla Leu Gly Ala 405 410 415 Thr Phe His Leu Leu Gln Ser Leu Gly Ile SerHis 420 425 <210> SEQ ID NO 8 <211> LENGTH: 9365 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: exon <222>LOCATION: (1)..(288) <221> NAME/KEY: exon <222> LOCATION: (1281)..(1580)<221> NAME/KEY: exon <222> LOCATION: (1820)..(1855) <221> NAME/KEY: exon<222> LOCATION: (2467)..(2555) <221> NAME/KEY: exon <222> LOCATION:(2863)..(2942) <221> NAME/KEY: exon <222> LOCATION: (3889)..(3950) <221>NAME/KEY: exon <222> LOCATION: (4894)..(4995) <221> NAME/KEY: exon <222>LOCATION: (5847)..(5987) <221> NAME/KEY: exon <222> LOCATION:(6966)..(7138) <221> NAME/KEY: exon <222> LOCATION: (8556)..(9365) <221>NAME/KEY: misc_feature <222> LOCATION: (3409) <223> OTHER INFORMATION: n= a or g or t or c <221> NAME/KEY: misc_feature <222> LOCATION: (9214)<223> OTHER INFORMATION: n = a or g or t or c <221> NAME/KEY:misc_feature <222> LOCATION: (9303) <223> OTHER INFORMATION: n = a or gor t or c <221> NAME/KEY: misc_feature <222> LOCATION: (9311) <223>OTHER INFORMATION: n = a or g or t or c <400> SEQUENCE: 8 gcctctgcaggtgtgcgagc aggattgctt ctgcaacaaa agcctccacc cagccacatc 60 ttgggaaaagaatggccact tcttggggca cagtcttttt catgctggtg gtatcctgtg 120 tttgcagcgctgtctcccac aggaaccagc agacttggtt tgagggtatc ttcctgtctt 180 ccatgtgccccatcaatgtc agcgccagca ccttgtatgg aattatgttt gatgcaggga 240 gcactggaactcgaattcat gtttacacct ttgtgcagaa aatgccaggt aagtgcaact 300 gggrcccttagtagagtctg taaatccaca ctttagcatc tcctcccaga aacaaatatg 360 ctgagagtttattatgtgaa ttacagaatc tcacacctag tggatgtctt tcttcagaga 420 actttggactacaattgaac atgtgggtta tttatttatt tttatttatt tgttttgttt 480 ttattttttaactttttttt tgagacaagg tcttgctttg ttgcccggtc tgtagtgcag 540 tggcatgatgacacatcact gcaaccttga cctcctgggc tcaagcagtc cttccacctc 600 agccccctgagttgttgaga ctacaggctt gtgccaccat gcccagctca tttttaaatt 660 tttttatagagacctgctca gactggcctc aaactcctag gctcaattga tcctcccacc 720 tcagcctcccaaagtactgg gattataggt gtaagtcacc atgcttggcc agaacacatg 780 gcttaattcaatgtgaaatt agaagagagc tgggctgtct gtagtctgaa acccatgtgt 840 tcaaaaagaatagttataat ttgttcttcc tctttaaaca tgggatactc cagggatcca 900 taatattcagaatatgggga gtggttttgg gagaaggatc acatgagaat ttcactgcca 960 tccttggacatgaggctagg aatccctgaa gattaacttt ttctgaattt gtcagtgttt 1020 tttcctcaggtcacttatgg agcctgggga aaggtggagg agttaggtgt ccaccagaga 1080 aatggtagcagaaatggacc ctcagaggtt gctctagtcc ttctttccag tactcctgca 1140 agacattcctcacaactagg atcattgggg taacttcagg gaagtcatag gaaaacttac 1200 agagacagagcccagcatct gaagcagcct aacttttggt aaccagctct ctcttctgtt 1260 ttgttccatgracaaaatag gacagcttcc aattctagaa ggggaagttt ttgattctgt 1320 gaagccaggactttctgctt ttgtagatca acctaagcag ggtgctgaga ccgttcaagg 1380 gctcttagaggtggccaaag actcaatccc ccgaagtcac tggaaaaaga ccccagtggt 1440 cctaaaggcaacagcaggac tacgcttact gccagaacac aaagccaagg ctctgctctt 1500 tgaggtaaaggagatcttca ggaagtcacc tttcctggta ccaaagggca gtgttagcat 1560 catggatggatccgacgaag gtgggagagg tgttgatatg cgttccaggg ggagaggggc 1620 aggatcagtgaaagatctaa ctaaaggaac tggggccagg aataaacaga aggaatgaga 1680 tagcaggaaatagaagacag ggagaaggga acatgtgctc tagacatgga atttagagag 1740 gaaaaaaaaaaaacaaggtt ggggccagga aagagaaaaa atgctctggg atctaatcct 1800 tgtctttctttctttttagg catattagct tgggttactg tgaattttct gacaggtaat 1860 acatcctcaagtttatcttt agagcttaac tagcttttac atgcatagtc agaggagtaa 1920 aagcctcttctttcattctg tattgtttct tcttctttaa aaaaggaaaa gaggctgggt 1980 gtggcagttcatgcctgtta attccagcgc tttgggaggc tgagttgggc agatcacttg 2040 aggccaggagttcaagacca gcctggccaa catggcgaaa ctccgtctct accaaaaata 2100 caaaaatagctgggcatggt ggtgtgtacc tgtagtccca gctactcagg aggctggaga 2160 atcacttgaacccaggaggc agaggttgca gtgagctgag agccgagatt gcgccactgc 2220 actccaggctggatgataga gcaagactct gtctccaaaa aggccttcca aaaaaaaaaa 2280 aaacacctgccttgaaggcc tctgctgcaa caagagtcct tccgagttga cattcacctg 2340 cagccttggggctggggagc agtggagtat atatggaata ccttcagtgt atgataagag 2400 caagagagacaagtgttggg ctgcccagga tgtcgaggct atttagagct ggctctcatt 2460 tgacaggtcagctgcatggc cacagacagg agactgtggg gaccttggac ctagggggag 2520 cctccacccaaatcacgttc ctgccccagt ttgaggtgag tcatttaatg aagatctggt 2580 tagaagtgcacttggcaggc gtatcatggt gccaagaaag aggcgcccca ttttcagcca 2640 gcagctctaccacgcttagg cagagtcaag tcaattaata actaggtgaa tgttcccttg 2700 ccatctcactgttcagaatc ccttcgtttc ctcaagccta gtgagattag ccccttaatc 2760 tgtcttcatctctgattttt tgctgggagg gacgggtggt ggtgtgaaca tcttcaggta 2820 attacagatcctgaatagct ttttgctttt tctgatttgc agaaaactct ggaacaaamt 2880 cyatrgggctacctcacttc ctttgagatg tttaacagca cttataagct ctatacacat 2940 aggtgaggacggggacaggg aagaagaata tttmwtkttg tatgatksty ytamctktss 3000 maagcwtkctcaaatctstk aytkyatctg attmgcaaaa acaaagdctg tgccaattcc 3060 ctaaggcctatcaactgaaa cccggwccac ttacaaagcc ggaggagcct aagaggcttc 3120 tccattcttggcctcaaaag cattaatata tgacttaaga gtcaaaagtt ttggstgggg 3180 cagtggcttcatgcctgtaa tccctgcact ttgggaggcc gaggtgggtg ggtcacctga 3240 ggtcaggcgtttragaccag cctggcaaac atggtgaaac cccgtctyta ctaaaataca 3300 aaaattagctggatatgaca gcgcacacct gtaatcctag ctattcagga ggctgaggca 3360 ggagaatcatttgaaccctg gaggcggaga ttgcagtgag ccgagatcnc accmctgcac 3420 ttcagccggagcgacagagc aagactcagt ctcaaaaaaa aaaaaaaaaa gaatcaaaag 3480 ctttctgtagggagaggaca cttcaagaag gctcaggcaa agctccttgc cagctccttt 3540 gagctggccttcagaggttc agaatccagc ctggaatgtg atcccagttg gggctaggag 3600 ctaagctaaagagagctttt ctgggaatgg ttcctagwgt gggaccctag gaattgtcac 3660 tgtctctggcctttgaatga taactgtggg gaattcttac tgcatagcct tgatccaaac 3720 tgtgcagaaattaccccttg ttgaccacag gagatgaata tgtcacagac agaacaaggt 3780 tttcatctttccagagggac acaggaacaa tgttactttt gaaagaggta gctttaggct 3840 agagaacttcaggaccagca tgaaattagt caatcctgta ttttacagtt acctgggatt 3900 tggattgaaagctgcaagac tagcaaccct gggagccctg gagacagaag gtttgtctgg 3960 gtacctgtgctgggggggga tggtgagggt gacacagata ctccgcttgc ttcttccctt 4020 ccttgatagccattctatgg aggaaaagat tatgttgaat tgggaggcaa atgttgtata 4080 atggacctaataatggcaaa ctccttttct agtttataag ttcagaagtt ttgatgtata 4140 ttattagccatttttagaat gaggtctact tgttcagggg taacagccta tgtctaggca 4200 gctgaagtgtctgcagaaat cccaggcttt acgaatacat tcagcaggag cttgctcaag 4260 ccctgagctttacattggag gcacaggaag cagagtctgt tctacatgca ggtggaacaa 4320 cagagtaactccattgatct cttcacaggt caggcagaac tgggttcagt cccagtgttg 4380 tgatatgaggcragtaacct atctgtgccc ctttcctcac attaaatgag aatttgcatt 4440 taaggcactttgtacagtaa tctgttattg ggatgacatc tattttgcat ttcagagtat 4500 acaaaacatcttcaagtata tttaattgaa gcctctcagc aaccagtgag gaaggtagca 4560 tagcatttctttcctgtttt tataaagggg aaagttgctg takgaaggtt ykrgatctct 4620 twragatgtgatraaagcca tggacccctc tgacaaaagc acatatgcat gaaaatttgc 4680 ttctggtttcagggggttca ccaaccccac aaagcctatc tttgaaccct gagttaagga 4740 ttcctgtcacaggatgttgt catggaatta atttcatagg attttaaggc ccagccccca 4800 tggtgaytcttttccacctc actggcttct tgcttgcctt cctccctctc tctcacttac 4860 ttacctcttaccttgtgccc tggattcttt cagggactga tgggcacact ttccggagtg 4920 cctgtttaccgagatggttg gaagcagagt ggatctttgg gggtgtgaaa taccagtatg 4980 gtggcaaccaagaaggcaag tgatgttttt tcactggtta aagttacgtt tacaatggaa 5040 gctctggaaaagtcccatgg gaaacttttt ccagaactca agagaagctt atcttgttgc 5100 agggasttattccaaagatc ttggcatgcc tccaaggact aatgtgaagt gacagtgaac 5160 aaagcagctgtcattctgca tcagccaagt gtcatggacc cattagatac ctgcccttag 5220 ccaagtgctgtggtgcacat ctattgtcct agctactcca aaggttgagg caagaggatc 5280 acttgagcccatgagttcaa ggctatagtg cgcaatgcca ctgcactcca gcctgggcaa 5340 cagggagaccctacctctta caaattaatt aagaagcata ttctaagcct aggtctaatg 5400 cagcagtgtgaaagcctgtt tagttaatgg ttagctattt aaattatagt aaaacttaaa 5460 accaagacaagaatgattca tcttcttata aaaggtatat acctgaatat caaggaatga 5520 acctgaattcccagtgaagg aagcaggcga gccctttagc tacttgctta caaatgctat 5580 ggaatgtaatgctaggcagc agcacaaggt tggccatgat ctggtgaata cagattaggc 5640 aggagagcggccatggagaa acagactggt gaggctgcag acgtttgctc atctttgttt 5700 tgacgcctcttgtcccaagc ctcagccttc tcctgctttc ttgaccttcc tgctgttccc 5760 tcattgtctccagcagcctg cctcagagag tgtccccttc ccccagcgtc gttctcacct 5820 tacccctgtgcacctttgcc tggcagggga ggtgggcttt gagccctgct atgccgaagt 5880 gctgagggtggtacgaggaa aacttcacca gccagaggag gtccagagag gttccttcta 5940 tgctttctcttactattatg accgagctgt tgacacagac atgattggtg agttcacccc 6000 aggtgtcagtccagagagga aggtggatag ggctgtggtg gggaaggtca aggagaaaga 6060 gcacttgaggtgctttgtcg gggtgattac ccacctcttt tctagtcact cgaacaaaag 6120 ggtggaaatgacttagagtc ttttggaggt gagagatgac caaaacaact atatgaggtc 6180 tttttttttttaacatgttt attgaggtat aattggcata caataagtgc cacatttaaa 6240 gtatacaatttaagttttgt catgtataca cccatgaatc catccagcac attgaagata 6300 ataaacatatttcaccacaa aaagtttcct cctgtctctt tataactttt cttcttatca 6360 caaaagcagtgtttttgcct aactgtgaaa gtatatgtac ctgatctgtc atggcctgag 6420 agagatgaattaatttccta ttattgtggg ggttttgttg ttgttgttgt tttggttttt 6480 tgtttgtttgtttgtttttt gagacagagt ctcactctgt tacccaggct ggagtgcaat 6540 ggcatgatctaggctcactg caacctctgc ctcccgggtt caaccgattc tcctgcccca 6600 gtctcctgagtagctgggat tacaggtgcc tgccaccaca cccggctaat ttttttttta 6660 atagagacgaggtttcacca tgttggtcag gctggtcttg aactcctgac ctcgttatct 6720 gccttcctcggcctcccaaa gtgctgggat tacaggcatg agccaccaca cccggcctat 6780 tgtgttttatgggtctgttt tttccattgt ggttaaatat acataacatg gaatagattg 6840 taaataagtaaattaggttg catagattac attatgtaca tgtgtatata atgaatgaat 6900 gaatgaatttccttatgctt ccttgaaggc gttttgatat cagataatct tctgttttat 6960 ttcagattatgaaaaggggg gtattttaaa agttgaagat tttgaaagaa aagccaggga 7020 agtgtgtgataacttggaaa acttcacctc aggcagtcct ttcctgtgca tggatctcag 7080 ctacatcacagccctgttaa aggatggctt tggctttgca gacagcacag tcttacaggt 7140 aagagacaggacaccagagt ctcataacag ccctcttttg tgggggttga gaaggagtaa 7200 gagcttgttcagtaatcaga gtagctagaa gtgaaattat gaggtatttt tgtttgggct 7260 atggacaaggtactgtgctg ggcaccatga atgtgggaaa ttatctcaat gcaatggtag 7320 cctccgagtgtattaccagg caagctatcg cacaggtcac agaacagaaa gactagcagc 7380 ccaaattaagatgccaagtc acatggttta tttatttatt tatttattta ttattatttt 7440 tttgagacggagtctygctc ttgttkccyr ggctggagtg cartggcryg atcwcrgctc 7500 actgcarcctycrcctcctg ggttcaagcg attctyctgc ctcagcctcc cragtagctg 7560 ggattacaggcrygcgccac cacgccyggc taattttttt gtatttttag tagagacggg 7620 gtttcaccatgttggccagg ctrktctyra actyctgayc tcaggtgatc cacccrcctc 7680 rgcctcccaaagtgctrgra ttayaggyrt gagccaccac kccyrgcctt ttttgktcgk 7740 ttctttttttttchtttttt tttttttttt gagacagggt cttgctctgt cacccatgct 7800 ggagtgcagtggcatgatct cagttcactg caacctctgc ctcccgggtt caagtgaccc 7860 tcccacctcagccctctgag tagctgggat tacaggtgtg tgccaccact cttgtctaat 7920 ttttttgtagagacggggtt ttgccatgtt gcccaggctg gtcttgaact cctggcctca 7980 agcaatccacctgccttggc ctcccaaagt gccaggagta caggcatgag ccactgcgcc 8040 tggccccatgtttggttatt attagtgctt aggaagaggc acttgcttac atagtaggag 8100 ttgagaagcttggtttgttc tttcctaccc ctagatctat tctcacctcc tgaccatgct 8160 ctttctgccacatctattat cattacaagt tgccttatct gaaattagtg aatcagaaaa 8220 taaagcaggggatactttgt gtagtttcaa cgttagggaa agttcagaat actgtctgtc 8280 taaactatctctctagaagg cctgatgggc cacaacctgg gccagaagca ttcagttcag 8340 atatgagaatggtgggtgta ggggcaatgg ccaatgggcc atggccggaa ggaaattgtt 8400 acagagtagtgggaagcctg caaagactgg cttctgtccg ttttgccttg gtttgcccat 8460 gtggatattctttgccaata ttttctgccc aagagctgtg cttgctagag ttggaaactg 8520 gatgaaaaggtgaagacttt ttttcttctc aacagctcac aaagaaagtg aacaacatag 8580 agacgggctgggccttgggg gccacctttc acctgttgca gtctctgggc atctcccatt 8640 gaggccacgtacttccttgg agacctgcat ttgccaacac ctttttaagg ggaggagaga 8700 gcacttagtttctgaactag tctggggaca tcctggactt gagcctagag atttaggttt 8760 aattaattttacacatctaa tagtgaactg ctgcctaacc actcaagagt acacagctgg 8820 caccagagcatcacagagag ccctgtgagc caaaaagtat agttttggaa cttaaccttg 8880 gagtgagagcccagggacag gtccctggaa accaaagaaa aatcgcattt caaccctttg 8940 agtgcctcattccactgaat atttaaattt tcctcttaaa tgggaaactg acttattgca 9000 atcccaagacccatcaatat cagtattttt ttcctcccta tacagggccc tgcccaccct 9060 tatctgcacccacctcccct gaaaaagaga gaaaaaaaaa aamccbggtt ttgctttccw 9120 tgtwtaatycamcgacmcaa aakgggacca tgtcaaaatc tgtwtgatcc tattytgggt 9180 tascyccaatcagccagctg aragccttcc taanttttaw taggatgara gagtaccycc 9240 taactgtgcataaattcagc cttaaaaaaa aaggcacccg ggctttgggg acatgtttgg 9300 gangggggggntgcctcata tacccacctt tggtttaata acattttatc agcactttgg 9360 gataa 9365<210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 9 gctacctcac ttcctttgag20 <210> SEQ ID NO 10 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 10 ctggctggtg aagttttcctc 21 <210> SEQ ID NO 11 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 11 gcaggtctcc aaggaagtacg 21 <210> SEQ ID NO 12 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 12 gtgagtgctc cctgcatctaacataattcc 30 <210> SEQ ID NO 13 <211> LENGTH: 45 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 13 gatgcagggagcactcacac tagtattcat gtttacacct ttgtg 45 <210> SEQ ID NO 14 <211>LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 14 gcgtagtcct gctgttgccc ctaggtacac tggggtctttttcc 44 <210> SEQ ID NO 15 <211> LENGTH: 31 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 15 gcaacagcaggactacgctt actgccagaa c 31 <210> SEQ ID NO 16 <211> LENGTH: 48 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:16 cccaagcgaa tatgccttcg tcttgtccag tcatgatgct aacactgc 48 <210> SEQ IDNO 17 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 17 cgaaggcata ttcgcttgggttactgtg 28 <210> SEQ ID NO 18 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 18 cttccttcactgggaattca gg 22 <210> SEQ ID NO 19 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:19 ctgtttaccg agatggttgg aagc 24 <210> SEQ ID NO 20 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: primer <400>SEQUENCE: 20 ttaaagcttg ggaaaagaat ggccacttc 29 <210> SEQ ID NO 21 <211>LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 21 agactcgagg tggctcaatg ggagatgcc 29 <210> SEQID NO 22 <211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 22 gcgctgtctc ccacagaggatcgcatcacc atcaccatca caaccagcag acttggtt 58 <210> SEQ ID NO 23 <211>LENGTH: 58 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 23 aaccaagtct gctggttgtg atggtgatgg tgatgcgatcctctgtggga gacagcgc 58 <210> SEQ ID NO 24 <211> LENGTH: 1601 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 gcgggctgccgcgcaagggt ggcgcgcgcg cgttttcctt gttcctggtc aacaaagaaa 60 tgtggagtgtcttggctgaa tcctcataca gacaagatca ttatggtgct gttaggttga 120 aaaagtgatataataaagga accaaggaga aaattcagaa ggaaagaaaa aattgcctct 180 gcaggtgtgcgagcaggatt gcttctgcaa caaaagcctc cacccagcca catcttggga 240 aaagaatggccacttcttgg ggcacagtct ttttcatgct ggtggtatcc tgtgtttgca 300 gcgctgtctcccacaggaac cagcagactt ggtttgaggg tatcttcctg tcttccatgt 360 gccccatcaatgtcagcgcc agcaccttgt atggaattat gtttgatgca gggagcactg 420 gaactcgaattcatgtttac acctttgtgc agaaaatgcc aggacagctt ccaattctag 480 aaggggaagtttttgattct gtgaagccag gactttctgc ttttgtagat caacctaagc 540 agggtgctgagaccgttcaa gggctcttag aggtggccaa agactcaatc ccccgaagtc 600 actggaaaaagaccccagtg gtcctaaagg caacagcagg actacgctta ctgccagaac 660 acaaagccaaggctctgctc tttgaggtaa aggagatctt caggaagtca cctttcctgg 720 taccaaagggcagtgttagc atcatggatg gatccgacga aggcatatta gcttgggtta 780 ctgtgaattttctgacaggt cagctgcatg gccacagaca ggagactgtg gggaccttgg 840 acctagggggagcctccacc caaatcacgt tcctgcccca gtttgagaaa actctggaac 900 aaactcctaggggctacctc acttcctttg agatgtttaa cagcacttat aagctctata 960 cacatagttacctgggattt ggattgaaag ctgcaagact agcaaccctg ggagccctgg 1020 agacagaagggactgatggg cacactttcc ggagtgcctg tttaccgaga tggttggaag 1080 cagagtggatctttgggggt gtgaaatacc agtatggtgg caaccaagaa ggggaggtgg 1140 gctttgagccctgctatgcc gaagtgctga gggtggtacg aggaaaactt caccagccag 1200 aggaggtccagagaggttcc ttctatgctt tctcttacta ttatgaccga gctgttgaca 1260 cagacatgattgattatgaa aaggggggta ttttaaaagt tgaagatttt gaaagaaaag 1320 ccagggaagtgtgtgataac ttggaaaact tcacctcagg cagtcctttc ctgtgcatgg 1380 atctcagctacatcacagcc ctgttaaagg atggctttgg ctttgcagac agcacagtct 1440 tacaggctgccgtactgagg tgatgggcca agctggagat atccccaaag cccatgttga 1500 caccctgtcctgcaagcgga tggactctgt gggctgcatc cctaagaata aagcagagtt 1560 caggtgtgacctctggcagc aaaaaaaaaa aaaaaaaaaa a 1601 <210> SEQ ID NO 25 <211> LENGTH:405 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 25 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 7580 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 9095 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys AlaLeu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe LeuVal Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp GluGly Ile Leu Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln LeuHis Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly GlyAla Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr LeuGlu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe AsnSer Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe GlyLeu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr GluGly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg TrpLeu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala GluVal Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr IleThr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr ValLeu Gln 385 390 395 400 Ala Ala Val Leu Arg 405 <210> SEQ ID NO 26 <211>LENGTH: 2762 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (148)..(1599) <400> SEQUENCE: 26gtggggtcgt atcccgcggg tggaggccgg ggtggcgccg gccggggcgg gggagcccaa 60aagaccggct gccgcctgct ccccggaaaa gggcactcgt ctccgtgggt gtggcggagc 120gcgcggtgca tggaatgggc tatgtga atg aaa aaa ggt atc cgt tat gaa act 174Met Lys Lys Gly Ile Arg Tyr Glu Thr 1 5 tcc aga aaa acg agc tac att tttcag cag ccg cag cac ggt cct tgg 222 Ser Arg Lys Thr Ser Tyr Ile Phe GlnGln Pro Gln His Gly Pro Trp 10 15 20 25 caa aca agg atg aga aaa ata tccaac cac ggg agc ctg cgg gtg gcg 270 Gln Thr Arg Met Arg Lys Ile Ser AsnHis Gly Ser Leu Arg Val Ala 30 35 40 aag gtg gca tac ccc ctg ggg ctg tgtgtg ggc gtg ttc atc tat gtt 318 Lys Val Ala Tyr Pro Leu Gly Leu Cys ValGly Val Phe Ile Tyr Val 45 50 55 gcc tac atc aag tgg cac cgg gcc acc gccacc cag gcc ttc ttc agc 366 Ala Tyr Ile Lys Trp His Arg Ala Thr Ala ThrGln Ala Phe Phe Ser 60 65 70 atc acc agg gca gcc ccg ggg gcc cgg tgg ggtcag cag gcc cac agc 414 Ile Thr Arg Ala Ala Pro Gly Ala Arg Trp Gly GlnGln Ala His Ser 75 80 85 ccc ctg ggg aca gct gca gac ggg cac gag gtc ttctac ggg atc atg 462 Pro Leu Gly Thr Ala Ala Asp Gly His Glu Val Phe TyrGly Ile Met 90 95 100 105 ttt gat gca gga agc act ggc acc cga gta cacgtc ttc cag ttc acc 510 Phe Asp Ala Gly Ser Thr Gly Thr Arg Val His ValPhe Gln Phe Thr 110 115 120 cgg ccc ccc aga gaa act ccc acg tta acc cacgaa acc ttc aaa gca 558 Arg Pro Pro Arg Glu Thr Pro Thr Leu Thr His GluThr Phe Lys Ala 125 130 135 gtg aag cca ggt ctt tct gcc tat gct gat gatgtt gaa aag agc gct 606 Val Lys Pro Gly Leu Ser Ala Tyr Ala Asp Asp ValGlu Lys Ser Ala 140 145 150 cag gga atc cgg gaa cta ctg gat gtt gct aaacag gac att ccg ttc 654 Gln Gly Ile Arg Glu Leu Leu Asp Val Ala Lys GlnAsp Ile Pro Phe 155 160 165 gac ttc tgg aag gcc acc cct ctg gtc ctc aaggcc aca gct ggc tta 702 Asp Phe Trp Lys Ala Thr Pro Leu Val Leu Lys AlaThr Ala Gly Leu 170 175 180 185 cgc ctg tta cct gga gaa aag gcc cag aagtta ctg cag aag gtg aaa 750 Arg Leu Leu Pro Gly Glu Lys Ala Gln Lys LeuLeu Gln Lys Val Lys 190 195 200 gaa gta ttt aaa gca tcg cct ttc ctt gtaggg gat gac tgt gtt tcc 798 Glu Val Phe Lys Ala Ser Pro Phe Leu Val GlyAsp Asp Cys Val Ser 205 210 215 atc atg aac gga aca gat gaa ggc gtt tcggcg tgg atc acc atc aac 846 Ile Met Asn Gly Thr Asp Glu Gly Val Ser AlaTrp Ile Thr Ile Asn 220 225 230 ttc ctg aca ggc agc ttg aaa act cca ggaggg agc agc gtg ggc atg 894 Phe Leu Thr Gly Ser Leu Lys Thr Pro Gly GlySer Ser Val Gly Met 235 240 245 ctg gac ttg ggc gga gga tcc act cag atcgcc ttc ctg cca cgc gtg 942 Leu Asp Leu Gly Gly Gly Ser Thr Gln Ile AlaPhe Leu Pro Arg Val 250 255 260 265 gag ggc acc ctg cag gcc tcc cca cccggc tac ctg acg gca ctg cgg 990 Glu Gly Thr Leu Gln Ala Ser Pro Pro GlyTyr Leu Thr Ala Leu Arg 270 275 280 atg ttt aac agg acc tac aag ctc tattcc tac agc tac ctc ggg ctc 1038 Met Phe Asn Arg Thr Tyr Lys Leu Tyr SerTyr Ser Tyr Leu Gly Leu 285 290 295 ggg ctg atg tcg gca cgc ctg gcg atcctg ggc ggc gtg gag ggg cag 1086 Gly Leu Met Ser Ala Arg Leu Ala Ile LeuGly Gly Val Glu Gly Gln 300 305 310 cct gct aag gat gga aag gag ttg gtcagc cct tgc ttg tct ccc agt 1134 Pro Ala Lys Asp Gly Lys Glu Leu Val SerPro Cys Leu Ser Pro Ser 315 320 325 ttc aaa gga gag tgg gaa cac gca gaagtc acg tac agg gtt tca ggg 1182 Phe Lys Gly Glu Trp Glu His Ala Glu ValThr Tyr Arg Val Ser Gly 330 335 340 345 cag aaa gca gcg gca agc ctg cacgag ctg tgt gct gcc aga gtg tca 1230 Gln Lys Ala Ala Ala Ser Leu His GluLeu Cys Ala Ala Arg Val Ser 350 355 360 gag gtc ctt caa aac aga gtg cacagg acg gag gaa gtg aag cat gtg 1278 Glu Val Leu Gln Asn Arg Val His ArgThr Glu Glu Val Lys His Val 365 370 375 gac ttc tat gct ttc tcc tac tattac gac ctt gca gct ggt gtg ggc 1326 Asp Phe Tyr Ala Phe Ser Tyr Tyr TyrAsp Leu Ala Ala Gly Val Gly 380 385 390 ctc ata gat gcg gag aag gga ggcagc ctg gtg gtg ggg gac ttc gag 1374 Leu Ile Asp Ala Glu Lys Gly Gly SerLeu Val Val Gly Asp Phe Glu 395 400 405 atc gca gcc aag tac gtg tgt cggacc ctg gag aca cag ccg cag agc 1422 Ile Ala Ala Lys Tyr Val Cys Arg ThrLeu Glu Thr Gln Pro Gln Ser 410 415 420 425 agc ccc ttc tca tgc atg gacctc acc tac gtc agc ctg cta ctc cag 1470 Ser Pro Phe Ser Cys Met Asp LeuThr Tyr Val Ser Leu Leu Leu Gln 430 435 440 gag ttc ggc ttt ccc agg agcaaa gtg ctg aag ctc act cgg aaa att 1518 Glu Phe Gly Phe Pro Arg Ser LysVal Leu Lys Leu Thr Arg Lys Ile 445 450 455 gac aat gtt gag acc agc tgggct ctg ggg gcc att ttt cat tac atc 1566 Asp Asn Val Glu Thr Ser Trp AlaLeu Gly Ala Ile Phe His Tyr Ile 460 465 470 gac tcc ctg aac aga cag aagagt cca gcc tca tagtggccga gccatccctg 1619 Asp Ser Leu Asn Arg Gln LysSer Pro Ala Ser 475 480 tccccgtcag cagtgtctgt gtgtctgcat aaaccctcctgtcctggacg tgacttcatc 1679 ctgaggagcc acagcacagg ccgtgctggc actttctgcacactggctct gggacttgca 1739 gaaggcctgg tgctgccctg gcatcagcct cttccagtcacatctggcca gagggctgtc 1799 tggacctggg ccctgctcaa tgccacctgt ctgcctgggctccaagtggg caggaccagg 1859 acagaaccac aggcacacac tgagggggca gtgtggctccctgcctgtcc catccccatg 1919 ccccgtccgc ggggctgtgg ctgctgctgt gcatgtccctgcgatgggag tcttgtctcc 1979 cagcctgtca gtttcctccc cagggcagag ctccccttcctgcaagagtc tgggaggcgg 2039 tgcaggctgt cctggctgct ctggggaagc cgagggacagccataacacc cccgggacag 2099 taggtctggg cggcaccact gggaactctg gacttgagtgtgtttgctct tccttgggta 2159 tgaatgtgtg agttcaccca gaggcctgct ctcctcacacattgtgtggt ttggggttaa 2219 tgatggaggg agacacctct tcatagacgg caggtgcccacctttcaggg agtctcccag 2279 catgggcgga tgccgggcat gagctgctgt aaactatttgtggctgtgct gcttgagtga 2339 cgtctctgtc gtgtgggtgc caagtgcttg tgtagaaactgtgttctgag cccccttttc 2399 tggacaccaa ctgtgtcctg tgaatgtatc gctactgtgagctgttcccg cctagccagg 2459 gccatgtctt aggtgcagct gtgccacggg tcagctgagccacagtccca gaaccaagct 2519 ctcggtgtct cgggccacca tccgcccacc tcgggctgaccccacctcct ccatggacag 2579 tgtgagcccc gggccgtgca tcctgctcag tgtggcgtcagtgtcggggc tgagcccctt 2639 gagctgcttc agtgaatgta cagtgcccgg cacgagctgaacctcatgtg ttccactccc 2699 aataaaaggt tgacaggggc ttctccttca aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2759 aaa 2762 <210> SEQ ID NO 27 <211> LENGTH: 484<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 Met LysLys Gly Ile Arg Tyr Glu Thr Ser Arg Lys Thr Ser Tyr Ile 1 5 10 15 PheGln Gln Pro Gln His Gly Pro Trp Gln Thr Arg Met Arg Lys Ile 20 25 30 SerAsn His Gly Ser Leu Arg Val Ala Lys Val Ala Tyr Pro Leu Gly 35 40 45 LeuCys Val Gly Val Phe Ile Tyr Val Ala Tyr Ile Lys Trp His Arg 50 55 60 AlaThr Ala Thr Gln Ala Phe Phe Ser Ile Thr Arg Ala Ala Pro Gly 65 70 75 80Ala Arg Trp Gly Gln Gln Ala His Ser Pro Leu Gly Thr Ala Ala Asp 85 90 95Gly His Glu Val Phe Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly 100 105110 Thr Arg Val His Val Phe Gln Phe Thr Arg Pro Pro Arg Glu Thr Pro 115120 125 Thr Leu Thr His Glu Thr Phe Lys Ala Val Lys Pro Gly Leu Ser Ala130 135 140 Tyr Ala Asp Asp Val Glu Lys Ser Ala Gln Gly Ile Arg Glu LeuLeu 145 150 155 160 Asp Val Ala Lys Gln Asp Ile Pro Phe Asp Phe Trp LysAla Thr Pro 165 170 175 Leu Val Leu Lys Ala Thr Ala Gly Leu Arg Leu LeuPro Gly Glu Lys 180 185 190 Ala Gln Lys Leu Leu Gln Lys Val Lys Glu ValPhe Lys Ala Ser Pro 195 200 205 Phe Leu Val Gly Asp Asp Cys Val Ser IleMet Asn Gly Thr Asp Glu 210 215 220 Gly Val Ser Ala Trp Ile Thr Ile AsnPhe Leu Thr Gly Ser Leu Lys 225 230 235 240 Thr Pro Gly Gly Ser Ser ValGly Met Leu Asp Leu Gly Gly Gly Ser 245 250 255 Thr Gln Ile Ala Phe LeuPro Arg Val Glu Gly Thr Leu Gln Ala Ser 260 265 270 Pro Pro Gly Tyr LeuThr Ala Leu Arg Met Phe Asn Arg Thr Tyr Lys 275 280 285 Leu Tyr Ser TyrSer Tyr Leu Gly Leu Gly Leu Met Ser Ala Arg Leu 290 295 300 Ala Ile LeuGly Gly Val Glu Gly Gln Pro Ala Lys Asp Gly Lys Glu 305 310 315 320 LeuVal Ser Pro Cys Leu Ser Pro Ser Phe Lys Gly Glu Trp Glu His 325 330 335Ala Glu Val Thr Tyr Arg Val Ser Gly Gln Lys Ala Ala Ala Ser Leu 340 345350 His Glu Leu Cys Ala Ala Arg Val Ser Glu Val Leu Gln Asn Arg Val 355360 365 His Arg Thr Glu Glu Val Lys His Val Asp Phe Tyr Ala Phe Ser Tyr370 375 380 Tyr Tyr Asp Leu Ala Ala Gly Val Gly Leu Ile Asp Ala Glu LysGly 385 390 395 400 Gly Ser Leu Val Val Gly Asp Phe Glu Ile Ala Ala LysTyr Val Cys 405 410 415 Arg Thr Leu Glu Thr Gln Pro Gln Ser Ser Pro PheSer Cys Met Asp 420 425 430 Leu Thr Tyr Val Ser Leu Leu Leu Gln Glu PheGly Phe Pro Arg Ser 435 440 445 Lys Val Leu Lys Leu Thr Arg Lys Ile AspAsn Val Glu Thr Ser Trp 450 455 460 Ala Leu Gly Ala Ile Phe His Tyr IleAsp Ser Leu Asn Arg Gln Lys 465 470 475 480 Ser Pro Ala Ser <210> SEQ IDNO 28 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 28 cgtatcccgc gggtggaggccggggtg 27 <210> SEQ ID NO 29 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 29 cttctgcaagtcccagagcc agtgtgc 27 <210> SEQ ID NO 30 <211> LENGTH: 21 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:30 ggagcccaaa agaccggctg c 21 <210> SEQ ID NO 31 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:31 tgaagtcacg tccaggacag g 21 <210> SEQ ID NO 32 <211> LENGTH: 36 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:32 cggaattcaa catgaaaaaa ggtaatccgt tatgaa 36 <210> SEQ ID NO 33 <211>LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 33 tgtctagatg aggctggact cttctg 26 <210> SEQ IDNO 34 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: oligonucleotide primer <400> SEQUENCE: 34atcctggact tgagcctaga g 21 <210> SEQ ID NO 35 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: oligonucleotide primer<400> SEQUENCE: 35 ctgatattga tgggtcttgg g 21 <210> SEQ ID NO 36 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:oligonucleotide primer <400> SEQUENCE: 36 ggatggaaag gagttggtca g 21<210> SEQ ID NO 37 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: oligonucleotide primer <400> SEQUENCE: 37gtccacatgc ttcacttcct c 21 <210> SEQ ID NO 38 <211> LENGTH: 502 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 38 Met Glu AspThr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10 15 Ile LeuAla Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu 20 25 30 Leu AlaVal Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45 Tyr GlyIle Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55 60 Tyr LysTrp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln 65 70 75 80 ValGlu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln 85 90 95 LysVal Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala 100 105 110Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu 115 120125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu 130135 140 Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro145 150 155 160 Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu GluGly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys PheSer Gln Lys 180 185 190 Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr AsnAsn Gln Glu Thr 195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser ThrGln Val Thr Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile Glu Ser Pro AspAsn Ala Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly Lys Asp Tyr Asn ValTyr Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln Ala Leu TrpGln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn Glu Ile LeuArg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val Val Asn ValSer Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe Glu Met ThrLeu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310 315 320 Asn TyrGln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser 325 330 335 TyrCys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro 340 345 350Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys 355 360365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu 370375 380 Met Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser385 390 395 400 Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys PheSer Gly 405 410 415 Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly Tyr His PheThr Ala Asp 420 425 430 Ser Trp Glu His Ile His Phe Ile Gly Lys Ile GlnGly Ser Asp Ala 435 440 445 Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu ThrAsn Met Ile Pro Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro Leu Ser HisSer Thr Tyr Val Phe Leu 465 470 475 480 Met Val Leu Phe Ser Leu Val LeuPhe Thr Val Ala Ile Ile Gly Leu 485 490 495 Leu Ile Phe His Lys Pro 500<210> SEQ ID NO 39 <211> LENGTH: 465 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 39 Met Ala Thr Ser Trp Gly Ala Val Phe Met LeuIle Ile Ala Cys Val 1 5 10 15 Gly Ser Thr Val Phe Tyr Arg Glu Gln GlnThr Trp Phe Glu Gly Val 20 25 30 Phe Leu Ser Ser Met Cys Pro Ile Asn ValSer Ala Gly Thr Phe Tyr 35 40 45 Gly Ile Met Phe Asp Ala Gly Ser Thr GlyThr Arg Ile His Val Tyr 50 55 60 Thr Phe Val Gln Lys Thr Ala Gly Gln LeuPro Phe Leu Glu Gly Glu 65 70 75 80 Ile Phe Asp Ser Val Lys Pro Gly LeuSer Ala Phe Val Asp Gln Pro 85 90 95 Lys Gln Gly Ala Glu Thr Val Gln GluLeu Leu Glu Val Ala Lys Asp 100 105 110 Ser Ile Pro Arg Ser His Trp GluArg Thr Pro Val Val Leu Lys Ala 115 120 125 Thr Ala Gly Leu Arg Leu LeuPro Glu Gln Lys Ala Gln Ala Leu Leu 130 135 140 Leu Glu Val Glu Glu IlePhe Lys Asn Ser Pro Phe Leu Val Pro Asp 145 150 155 160 Gly Ser Val SerIle Met Asp Gly Ser Tyr Glu Gly Ile Leu Ala Trp 165 170 175 Val Thr ValAsn Phe Leu Thr Gly Gln Leu His Gly Arg Gly Gln Glu 180 185 190 Thr ValGly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr Phe 195 200 205 LeuPro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr Leu 210 215 220Thr Ser Phe Glu Met Phe Asn Ser Thr Phe Lys Leu Tyr Thr His Ser 225 230235 240 Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly Ala245 250 255 Leu Glu Ala Lys Gly Thr Asp Gly His Thr Phe Arg Ser Ala CysLeu 260 265 270 Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val LysTyr Gln 275 280 285 Tyr Gly Gly Asn Gln Glu Gly Glu Met Gly Phe Glu ProCys Tyr Ala 290 295 300 Glu Val Leu Arg Val Val Gln Gly Lys Leu His GlnPro Glu Glu Val 305 310 315 320 Arg Gly Ser Ala Phe Tyr Ala Phe Ser TyrTyr Tyr Asp Arg Ala Ala 325 330 335 Asp Thr His Leu Ile Asp Tyr Glu LysGly Gly Val Leu Lys Val Glu 340 345 350 Asp Phe Glu Arg Lys Ala Arg GluVal Cys Asp Asn Leu Gly Ser Phe 355 360 365 Ser Ser Gly Ser Pro Phe LeuCys Met Asp Leu Thr Tyr Ile Thr Ala 370 375 380 Leu Leu Lys Asp Gly LeuGly Phe Ala Glu Arg His Pro Leu Thr Ala 385 390 395 400 His Lys Glu SerGlu Gln His Arg Asp Trp Leu Gly Leu Gly Gly His 405 410 415 Leu Ser ProAla Pro Val Ser Gly His His Gln Leu Arg Pro Ser Ser 420 425 430 Thr SerGlu Ala Cys Ile Ser Glu Pro Val Phe Ser Gln Glu Gly Val 435 440 445 AspSer Glu Thr Phe Ser Asp Leu Ser Gly Lys Ala Trp Pro Glu Thr 450 455 460Arg 465

What is claimed is:
 1. An isolated polynucleotide encoding an apyraseand/or NDPase and comprising a nucleotide sequence having at least about80% sequence identity to a human polynucleotide selected from the groupconsisting of: (a) a polynucleotide having the nucleotide sequence ofSEQ ID NO. 2; and (b) a polynucleotide having the protein codingnucleotide sequence of the polynucleotide sequence of (a).
 2. Anisolated polynucleotide encoding an apyrase and comprising a nucleotidesequence having at least about 90% sequence identity to a polynucleotideselected from the group consisting of: (a) a polynucleotide having thenucleotide sequence of SEQ ID NO. 2; and (b) a polynucleotide having theprotein coding nucleotide sequence of the polynucleotide sequence of(a).
 3. An isolated polynucleotide encoding a polypeptide with apyraseand/or NDPase activity, comprising a polynucleotide selected from thegroup consisting of: (a) polynucleotides that encode the mature proteincoding amino acid sequence of SEQ ID NO.
 3. 4. An isolatedpolynucleotide encoding a polypeptide with apyrase and/or NDPaseactivity that hybridizes under stringent conditions to the complement ofa polynucleotide of SEQ ID NO.
 2. 5. The polynucleotide of any one ofclaims 1 through 4 which is a DNA.
 6. The DNA of claim 5 which is awholly or partially chemically synthesized DNA molecule.
 7. Ananti-sense polynucleotide which specifically hybridizes with thecomplement of the polynucleotide of claim
 4. 8. The polynucleotide ofclaim 1 which comprises the nucleotide sequence of SEQ ID NO. 2 or 24 orthe mature protein coding portions thereof.
 9. An isolatedpolynucleotide which comprises a complement of the polynucleotide ofclaim
 1. 10. An expression vector comprising the DNA of claim
 5. 11. Ahost cell comprising the DNA of claim
 5. 12. A host cell geneticallyengineered to express the DNA of claim
 5. 13. An isolated humanpolypeptide with apyrase and/or NDPase activity comprising: (a) themature protein coding sequence of SEQ ID NO. 3; or (b) an amino acidsequence having at least about 80% sequence identity to SEQ ID NO. 3.14. An isolated polypeptide with apyrase and/or NDPase activitycomprising: (a) the CD39-like protein coding sequence of SEQ ID NO. 3;or (b) an amino acid sequence having at least about 90% sequenceidentity to SEQ ID NO.
 3. 15. The polypeptide of claim 13 or 14 whichcomprises the amino acid sequence of SEQ ID NO. 3 or 25 or the matureprotein portions thereof.
 16. The polypeptide of claim 13 or 14 whereinthe polypeptide comprises at least one amino acid substitution selectedfrom the group consisting of: D168→T, S170→Q and L175→F.
 17. Thepolypeptide of claim 16 comprising a polypeptide having the amino acidsequence set forth in SEQ ID NO.
 7. 18. A method for producing aCD39-like polypeptide comprising the steps of: (a) growing a culture ofcells according to claim 11 under conditions permitting expression of aCD39-like polypeptide; and (b) isolating the CD39-like polypeptide fromthe host cell or its growth medium.
 19. A composition comprising thepolypeptide of claim 13, 14 or 15 and a pharmaceutically acceptablecarrier.
 20. An antibody specifically immunoreactive with a polypeptideencoded by the polynucleotide according to claim
 1. 21. The antibodyaccording to claim 20 which is a monoclonal antibody.
 22. A hybridomawhich secretes the antibody according to claim
 21. 23. An anti-idiotypeantibody specifically immunoreactive with the antibody according toclaim
 21. 24. A method for detecting a polynucleotide of claim 1, 2 or 3in a sample comprising the steps of: (a) contacting the sample with acompound that binds to and forms a complex with the polynucleotide for aperiod sufficient to detect the complex; and (b) detecting the complexso that if a complex is detected, a polynucleotide of claim 1, 2 or 3 isdetected.
 25. A method for detecting a polynucleotide of claim 1, 2 or 3in a sample comprising the steps of: (a) contacting the sample understringent hybridization conditions with nucleic acid primers that annealto a polynucleotide of claim 1, 2 or 3 under such conditions; and (b)amplifying the polynucleotides of claim 1, 2 or 3 so that if apolynucleotide is amplified, a polynucleotide of claim 1, 2 or 3 isdetected.
 26. The method of claim 25 wherein the polynucleotide is anRNA molecule that encodes a polypeptide of claim 13 or 14, and themethod further comprises reverse transcribing an annealed RNA moleculeinto a cDNA polynucleotide.
 27. A method for detecting a polypeptide ofclaim 13 or 14 in a sample comprising: (a) contacting the sample with acompound that binds to and forms a complex with the polypeptide for aperiod sufficient to detect the complex; and (b) detecting the complexso that if a complex is detected, a polypeptide of claim 13 or 14 isdetected.
 28. A method for identifying a compound that binds to apolypeptide of claim 13 or 14 comprising: (a) contacting a compound witha polypeptide of claim 13 or 14 for a time sufficient to form apolypeptide/compound complex; and (b) detecting the complex so that if apolypeptide/compound complex is detected, a compound that binds to apolypeptide of claim 13 or 14 is detected.
 29. A method for identifyinga compound that binds to a polypeptide of claim 13 or 14 comprising: (a)contacting a compound with a polypeptide of claim 13 or 14, in a cell,for a time sufficient to form a polypeptide/compound complex, whereinthe complex drives expression of a reporter gene sequence in the cell,and (b) detecting the complex by detecting reporter gene sequenceexpression so that if a polypeptide/compound complex is detected, acompound that binds to a polypeptide of claim 13 or 14 is identified.30. A method of identifying a modulator compound of a CD39-likepolypeptide with apyrase activity comprising the steps of: (a)contacting the CD39-like polypeptide encoded by the polynucleotide ofclaim 1, 2 or 3 with a substrate in the presence and absence of a testcompound; (b) comparing apyrase activity of the CD39-like polypeptide inthe presence and absence of the test compound; and (c) identifying thetest compound as a modulator compound when biological activity of theCD39-like polypeptide is increased or decreased in the presence of thetest compound.
 31. A method of identifying a modulator compound of aCD39-like polypeptide with NDPase activity comprising the steps of: (a)contacting the CD39-like polypeptide encoded by the polynucleotide ofclaim 1, 2 or 3 with a substrate in the presence and absence of a testcompound; (b) comparing NDPase activity of the CD39-like polypeptide inthe presence and absence of the test compound; and (c) identifying thetest compound as a modulator compound when biological activity of theCD39-like polypeptide is increased or decreased in the presence of thetest compound.
 32. A chimeric polypeptide comprising one or more domainsof a CD39-like polypeptide fused to one or more domains of heterologouspeptide or polypeptide, e.g., an immunoglobulin constant region.
 33. Amethod of treatment comprising administering to a mammalian subject inneed thereof a therapeutic amount of a composition comprising apolypeptide of claim 13 or 14 and a pharmaceutically acceptable carrier.34. A method of treatment comprising administering to a mammaliansubject in need thereof a therapeutic amount of a composition comprisingan antibody that specifically binds to a polypeptide of claim 13 or 14and a pharmaceutically acceptable carrier.
 35. A method of inhibitingplatelet function comprising administering the polypeptide of claim 13or 14 to a medium comprising platelets.
 36. A method of treatingthrombotic diseases comprising administering a therapeutic amount of thepolypeptide of claim 13 or 14 to a mammalian subject in need thereof.37. A method of hydrolyzing nucleotidediphosphates comprisingadministering the polypeptide of claim 13 or 14 to a medium comprisingnucleotidediphospates.
 38. A method of inhibiting platelet aggregationin a mammalian subject comprising the step of reducing the ratio ofADP:ATP in said mammalian subject to a less than normal ratio.
 39. Themethod of claim 38 wherein said ratio is reduced by administration ofCD39-L4 having the sequence set forth in SEQ ID NO: 3 or a polypeptidehaving NDPase activity and at least about 90% sequence identity to SEQID NO:
 3. 40. The method of claim 38 wherein said ratio is reduced byadministration of CD39-L66 having the sequence set forth in SEQ ID NO:25 or a polypeptide having NDPase activity and at least about 90%sequence identity to SEQ ID NO:
 25. 41. The method of claim 38 whereinsaid ratio is reduced by administration of CD39-L2 having the sequenceset forth in SEQ ID NO: 27 or a polypeptide having NDPase activity andat least about 90% sequence identity to SEQ ID NO:
 27. 42. The method ofclaim 38 - 41 wherein the ratio of ADP:ATP is reduced systemically incirculation.
 43. The method of claim 38 - 41 wherein the ratio ofADP:ATP is reduced locally within an area selected from the groupconsisting of heart, brain, kidney, lung and limbs.
 44. The method ofclaim 38-41 wherein the ratio of ADP:ATP is reduced withoutsignificantly affecting ATP levels.
 45. A method of identifying acompound capable of reducing the ratio of ADP:ATP to a less than normalratio comprising the steps of: (a) determining apyrase activity of saidcompound on ATP; (b) determining apyrase activity of said compounds onADP; and (c) selecting a compound that has greater activity with respectto ADP compared to ATP.